Target and method for inhibition of bacterial RNA polymerase

ABSTRACT

Target and method for inhibition of bacterial RNA polymerase disclosed are targets and methods for specific binding and inhibition of RNAP from bacterial species.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/257,801, filed Oct. 24, 2008 now U.S. Pat. No. 8,198,021, which is acontinuation of Ser. No. 10/526,323, filed Oct. 3, 2005 now abandoned,which is a National Phase of Application No. PCT/US03/27457, filed Sep.4, 2003, which claims priority from 60/474,608, filed Jun. 2, 2003;60/474,607, filed Jun. 2, 2003; and 60/407,684, filed Sep. 4, 2002, thedisclosures of which are all hereby incorporated by reference herein.

GOVERNMENT SUPPORT

This invention, was supported with U.S. Government funds (NIHRO1-GM41376). Therefore, the Government may have rights in theinvention.

BACKGROUND ART

Bacterial infections remain among the most common and deadly causes ofhuman disease. Infectious diseases are the third leading cause of deathin the United States and the leading cause of death worldwide (Binder etal., Science 284: 1311-1313 (1999). Multi-drug-resistant bacteria nowcause infection that poses a grave and growing threat to public health.It has been shown that bacterial pathogens can acquire resistance tofirst-line and even second-line antibiotics. (See, Stuart B. Levy, TheChallenge of Antibiotic Resistance, in Scientific American, 46-53(March, 1998); Walsh, C. (2000) Nature 406, 775-781; Schluger, M. (2000)Int. J. Tuberculosis Lung Disease 4, S71-S75; Raviglione et al., (2001)Ann NY Acad Sci 953, 88-97). New approaches to drug development arenecessary to combat the ever-increasing number of antibiotic-resistantpathogens.

RNA is synthesized in cellular organisms by a complex molecular machine,known as RNA polymerase (“RNAP”). In its simplest bacterial form, RNAPcomprises at least 4 subunits with a total molecular mass of around 400kDa. RNAP mediates the transcription of DNA to produce RNA. BacterialRNAP is a multimeric protein consisting of subunits α₂, β, β′, and ω. Anα factor is required for initiation of transcription by forming aholoenzyme complex.

Currently, there are a few known antibiotics that target RNAP—notably,rifampicin and rifampicin analogs (See Mitchison, D. (2000) Int. J.Tuberculosis Lung Disease 4, 796-806). Rifampicin is the onlyanti-tuberculosis compound able to rapidly clear infection and preventrelapse. Without rifampicin, treatment lengths must increase from 6months to at least 18 months to ensure prevention of relapse. Rifampicinacts by specifically inhibiting RNAP (Campbell et al., (2001) Cell 104,901-912). Rifampicin binds to a site adjacent to the active center ofbacterial RNAP, the exit channel, and physically prevents synthesis ofproducts longer than ˜4 nucleotides. Unfortunately, tuberculosis strainsresistant to rifampicin (and rifampicin analogs) are becomingwidespread, effectively removing rifampicin from the therapeuticarsenal. Thus, there is a need for novel antibiotics that target thesame bacterial enzyme as rifampicin, RNAP, (and thus that have the samebiochemical and therapeutic effects as rifampicin). There is also a needto develop methods for identifying antibiotics that interfere withbacterial RNAP.

Recently crystallographic structures have been determined for bacterialRNAP and eukaryotic RNAP II, and, based on the crystallographicstructures, biophysical results, and biochemical results, models havebeen proposed for the structures of transcription initiation andelongation complexes (Gnatt et al., (2001) Science 292, 1876-1882;Ebright, R. (2000) J. Mol. Biol. 304, 687-689; Naryshkin et al., (2000Cell 101, 601-611; Kim et al., (2000) Science 288, 1418-1421; Korzhevaet al., (2000) Science 289, 619-625; and Mekler et al., (2002) Cell108:599-614). The models propose that nucleic acids completely fill theactive-center cleft of RNAP, such that the only route by which incomingnucleoside triphosphate substrates (NTPs) can access the active centeris through an approximately 25 Å long, 10 Å wide tunnel known as the“secondary channel” or “pore,” that bores through the floor of theactive-center cleft of RNAP opposite the active-center cleft. (Gnatt etal., (2001 Science 292, 1876-1882; Ebright, R. (2000) J. Mol. Biol. 304,687-639).

SUMMARY OF THE INVENTION

Applicant has discovered that a region within the secondary channelcomprising two adjacent short peptide segments of the RNAP β′ subunitare conserved in amino-acid sequence in bacterial species, includingboth Gram-positive bacteria and Gram-negative bacteria. Throughout thefollowing specification, this region is referred to as the “β′ pocket”or the “target,” and the two short peptide segments are referred to as“homologous secondary channel amino acids or amino acid sequences.”Applicant has also discovered that this same region is not conserved,and in fact is radically different, in amino-acid sequence in eukaryoticRNAP, such as human RNAP I, RNAP II, and RNAP III.

Accordingly, a first aspect of the present invention is directed to amethod for identifying agents that bind to a homologous RNAP secondarychannel amino acid sequence, comprising preparing a reaction solutioncomprising the agent to be tested and an entity containing a homologoussecondary channel amino acid sequence; and detecting presence or amountof binding. In a preferred embodiment, detection or quantitation ofbinding is conducted relative to binding of the agent to an entitycontaining an altered homologous amino acid sequence. In other preferredembodiments, quantitation of binding is compared to binding of the21-amino acid (GGAGHVPEYFVGOGTPISFYG) (SEQ ID NO:1) bacteriocidalpeptide microcin J25 (“MccJ25”), or a derivative thereof, to the entityvia the homologous secondary channel amino acid sequence.

Another aspect of the present invention is directed to a method foridentifying agents that inhibit an activity of RNAP via binding to ahomologous secondary channel amino acid sequence. This aspect entailspreparing a reaction solution comprising the agent to be tested, acatalytic entity containing a homologous secondary channel amino acidsequence, and a substrate for the entity; and determining extent ofinhibition of RNAP activity via binding of the agent to the homologoussecondary channel amino acid sequence.

In some preferred embodiments, quantitation of inhibition is compared tobinding of MccJ25 to the catalytic entity via the homologous secondarychannel amino acid sequence. MccJ25 is active against Gram-negativebacteria (Delgado et al., (2001) J. Bacteriol. 183:4543-4550). It wasrecently discovered that RNAP is a target of MccJ25. (Delgado et al.,(2001) J. Bacteriol. 183:4543-4550; Yuzenkova et al., (2002) J. Biol.Chem. 277: 50867-50875). The present invention provides that analogs ofMccJ25 inhibit transcription by requiring determinants within the β′pocket. The invention also provides probe-labeled derivatives of MccJ25,which can be used for detailed analysis of MccJ25 and otherβ′-pocket-directed inhibitors of RNAP. In one aspect of the invention,probe-labeled derivatives of MccJ25 can be used in high-throughputscreening, by assessing ability of compounds or compound mixtures tocompete with a probe-labeled derivative of MccJ25 for binding to RNAP oran RNAP fragment containing the target. Thus, the invention has broadapplications in analysis of RNAP structure and function, control ofbacterial gene expression, control of bacterial growth, antibacterialchemistry, antibacterial therapy, and drug discovery.

A further aspect of Applicants' invention is directed to an analog ofMccJ25 having an amino acid sequence that differs from MccJ25 in termsof at least one amino acid deletion, insertion, or substitution, andthat binds bacterial RNAP or inhibits bacterial RNAP catalytic activityto a greater extent than MccJ25.

The present invention also provides for the identification of potentialantibacterial agents or antibiotics that, because of their bindingaffinity to regions within RNAP that are conserved among bacteria, havebroad-spectrum activity. It also provides for the identification ofpotential anti-bacterial agents or antibiotics that, because of theirsubstantial lack of binding affinity for eukaryotic RNAPs, will berelatively non-disruptive to normal cellular functions of the host.

It is anticipated that compounds identified according to the target andmethod of this invention would have applications not only inantibacterial therapy, but also in: (a) identification of bacterial RNAP(diagnostics, environmental-monitoring, and sensors applications), (b)labeling of bacterial RNAP (diagnostics, environmental-monitoring,imaging, and sensors applications), (c) immobilization of bacterial RNAP(diagnostics, environmental-monitoring, and sensors applications), (d)purification of bacterial RNA polymerase (biotechnology applications),(e) regulation of bacterial gene expression (biotechnologyapplications), and (f) antisepsis (antiseptics, disinfectants, andadvanced-materials applications).

These and other aspects of the present invention will be betterappreciated by reference to the following drawings and DetailedDescription.

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a sequence alignment for the target amino acidresidues 736-747 and 779-781 of the a′ subunit of RNAP from Escherichiacoli (SEQ ID NO: 2); and corresponding residues of the P′ subunits ofHaemophilus influenzae (SEQ ID NO: 3), Vibrio cholera (SEQ ID NO: 4),Pseudomonas aeruginose (SEQ ID NO: 5), Treponema pallidum (SEQ ID NO:6), Borrelia burgdorferi (SEQ ID NO: 7), Xyella fastidiosa (SEQ ID NO:8), Camploacter jejuni (SEQ ID NO: 9), Neisseria meningitides (SEQ IDNO: 10), Rickettsia prowazekii (SEQ ID NO: 11, Thermotoga maritime (SEQID NO: 12), Chlamydia trachomatis (SEQ ID NO: 13), Mycoplasma pneumoniae(SEQ ID NO: 14), Bacillus subtilis (SEQ ID NO: 15), Staphylococcusaureus (SEQ ID NO: 16), Mycobacterium tuberculosis (SEQ ID NO: 17),Synechocystis sp. (SEQ ID NO: 18), Aquifex aeolicus (SEQ ID NO: 19),Deinococcus radiodurans (SEQ ID NO: 20), Thermus thermophilus (SEQ IDNO: 21), and Thermus aquaticus (SEQ ID NO: 22) (collectively, “thehomologous bacterial RNAP secondary channel amino acids”); andcorresponding residues of the largest subunits of human RNAP I (SEQ IDNO: 23), RNAP II (SEQ ID NO: 24) and RNAP III (SEQ ID NO: 25)).

FIG. 2 illustrates a model showing the location of the target within thestructure of RNAP.

FIG. 3 illustrates that MccJ25 does not inhibit open-complex formation.Results of electrophoretic mobility shift experiments assessing effectsof MccJ25 on open-complex formation. RP_(o), RNAP-promoter open complex;P, free promoter DNA.

FIG. 4. illustrates that MccJ25 inhibits abortive initiation andelongation. (A) Results of transcription experiments assessing effectsof MccJ25 on abortive initiation and elongation. (B) As (A), but withRNAP derivative bearing Thr931→Ile substitution in RNAP β′ subunit (seeDelgado et al., 2001).

FIG. 5 illustrates that MccJ25 inhibits at the level of NTP uptake. (A)Results of transcription experiments assessingNTP-concentration-dependence of effects of MccJ25 on abortive initiationand elongation. (B) Double-reciprocal (Lineweaver-Burk) plot forinhibition of synthesis of 7-mer and 8-mer abortive products. Filledcircles, no MccJ25; open circles, 1 μM MccJ25; filled triangles, 10 μMMccJ25; open triangles, 100 μM MccJ25. Data are from (A). Lines are fitsto a partial-competitive model of inhibition (K_(i)=1.4±0.2 μM; α=15±3;r²=0.99). (C) Double-reciprocal (Lineweaver-Burk) plot for inhibition ofsynthesis of 3-mer and 4-mer abortive products. Filled circles, noMccJ25; open circles, 1 μM MccJ25; filled triangles, 10 μM MccJ25; opentriangles, 100 μM MccJ25. Data are from fluorescence-detected abortiveinitiation assays (see Experimental Procedures). Lines are fits to apartial-competitive model of inhibition (K_(i)=1.2±0.3 μM; α=87±2;r²=0.97).

FIG. 6 illustrates that MccJ25 binds within the RNAP secondary channel.Schematic map of RNAP β′ subunit showing locations of conserved regions(conserved regions A-H lettered and shaded; additional conserved regionslightly shaded) and locations of substitutions that conferMccJ25-resistance (substitution of Delgado et al., 2001 in black;substitutions from random mutagenesis as red bars; and substitutionsfrom saturation mutagenesis in pink). (B)-(D) Three-dimensionalstructure of RNAP showing locations of substitutions that conferMccJ25-resistance [substitution of Delgado et al., 2001 in red in (B);substitutions from random mutagenesis in red in (C); and substitutionsfrom random and saturation mutagenesis in red in (D)]. Each panelpresents a stereodiagram. In each panel, the view is directly into theRNAP secondary channel—toward the active-center Mg⁺⁺ (white sphere atcenter). Atomic coordinates are based on the crystallographic structureof Thermus thermophilics RNAP at 2.6 Å resolution (Vassylyev et al.,(2002) Nature 417, 712-719) PDB accession 1IW7; σ subunit omitted forclarity). Correspondences between residues of E. coli RNAP β′ and T.thermophilics RNAP β′ are based on a comprehensive sequence alignment ofbacterial RNAP β′ subunits, archaeal RNAP A subunits, and eukaryoticRNAP largest subunits (unpublished).

BEST MODE OF CARRYING OUT THE INVENTION

The present invention provides methods of designing specific inhibitorsof bacterial RNAP, the enzyme responsible for transcription. The presentinvention also provides for the design of small-molecule inhibitors(i.e., diameter <20 Å, MW≦2.1 kDa) that bind, with a k_(d) 21 1 μM, to atarget site that is highly conserved in bacterial RNAP, but distinctlydifferent in eukaryotic RNAP. The invention provides targets and methodsfor specific binding and inhibition of RNAP from bacterial species. Theinvention has applications in control of bacterial gene expression,control of bacterial growth, antibacterial chemistry, and antibacterialtherapy.

Recently, crystallographic structures have been determined for bacterialRNAP and eukaryotic RNAP II (Zhang et al., (1999) Cell 98, 811-824;Cramer et al., (2000) Science 288, 640-649; Cramer et al., (2001)Science 292, 1863-1876; Gnatt et al., (2001 Science 292, 1876-1882; andEbright, R. (2000) J. Mol. Biol. 304, 687-689), and, based on thecrystallographic structures, biophysical results, and biochemicalresults, models have been proposed for the structures of transcriptioninitiation and elongation complexes (Gnatt et al., (2001) Science 292,1876-1882; Ebright, R. (2000) J. Mol. Biol. 304, 687-689; Naryshkin etal., (2000 Cell 101, 601-611; Kim et al., (2000) Science 288, 1418-1421;Korzheva et al., (2000) Science 289, 619-625; and Mekler et al., (2002)Cell 108:599-614).

The models for the transcription elongation complex imply that nucleicacids completely fill the active-center cleft of RNAP, and thus the onlyroute by which incoming nucleoside-triphosphate substrates (“NTPs”) canaccess the active center is through a ˜25 Å long, ˜10 Å wide tunnel—the“secondary channel” or “pore”—that bores through the floor of theactive-center cleft of RNAP and extends to the exterior surface of RNAPopposite the active-center cleft (Gnatt et al., (2001) Science 292,1876-1882; Ebright, R. (2000) J. Mol. Biol. 304, 687-689; and Korzhevaet al., (2000) Science 289, 619-625).

The models for the transcription elongation complex imply that the RNAPsecondary channel mediates multiple biochemical activities important forfunction of RNAP, including: uptake of NTPs, release of pyrophosphateproduct, release of abortive-RNA and edited-RNA products, interactionwith RNA product during transcriptional pausing, interaction with RNAproduct during transcriptional arrest, interaction with RNA productduring editing, and interaction with the elongation factors GreA andGreB.

It has now been found, and is disclosed herein, that physically blockingthe RNAP secondary channel with a small molecule inhibits at least oneof these activities. Specifically, it has now been found, and isdisclosed herein, that physically blocking the RNAP secondary channelwith a small molecule prevents uptake of NTPs by RNAP and thus inhibitstranscription.

The present invention is based in part on Applicant's discovery that aregion within the RNAP secondary channel comprising amino acids 736-747and 779-781 of the RNAP β′ subunit from Escherichia coli (the “β′pocket” or “target”) is a useful target for compounds that blocktranscription. It was found that said pocket is lined with residues thatare invariant or nearly invariant in RNAP from bacterial species, butthat are radically different in RNAP from eukaryotic species (FIG. 1).It further was found that this region forms a ˜5 Å shallow pocket withinthe wall of the RNAP secondary channel (FIG. 2).

Models for the transcription elongation complex suggest that nucleicacids completely fill the active channel of RNAP, and thus that the onlyroute by which incoming nucleoside-triphosphate (NTP) substrates canaccess this active center is through this secondary channel. The presentinvention therefore relates to molecules that bind to the secondarychannel of RNAP from Escherichia coli, or in corresponding regions ofRNAP from other bacterial species. In a preferred embodiment, thepresent invention relates to molecules that bind to RNAP in thesecondary channel of RNAP from Escherichia coli, or in correspondingregions of RNAP from other bacterial species and prevent RNAP fromcarrying out at least one biochemical activity with which the secondarychannel is associated (e.g., uptake of NTP substrates, release ofpyrophosphate product, release of edited nucleotide and oligonucleotideproducts, interaction with RNA product during transcriptional pausing,interaction with RNA product during transcriptional arrest, interactionwith RNA product during editing, and interaction with the elongationfactors GreA and GreB). In one aspect of the present invention, methodsand compositions are described involving compounds that specificallyblock the secondary channel of RNAP resulting in inhibition oftranscription.

The target referred to above in Escherichia coli is similar in aminoacid sequence to that of most or all other species of bacterial RNAP andis called herein the homologous bacterial RNAP secondary channel (FIG.1). (For example, amino acid residues 736-747 and 779-781 of the β′subunit of RNAP from Escherichia coli exhibit high similarity to aminoacid residues 740-751 and 783-785 of the β′ subunit of RNAP fromBacillus subtilis (FIG. 1.) Thus, the discovery of a molecule that bindsto the target and inhibits an activity associated with the secondarychannel in Escherichia coli RNAP also is likely to bind to the target aninhibit an activity associated with the secondary channel in otherspecies of bacterial RNAP. Therefore, molecules found to be haveantibiotic activity (through binding to the target and inhibiting anactivity associated with the secondary channel) against Escherichia coliare likely to be found to have antibiotic activity against otherbacterial species.

In contrast, the target differs radically in amino acid sequence betweenbacterial RNAP and eukaryotic RNAP, including human RNAP I, RNAP II, andRNAP III (FIG. 1). This allows for the identification of molecules thatbind, in a target-dependent fashion, to bacterial RNAP, but that do notbind, or that bind substantially less well, to eukaryotic RNAP. Thisalso allows for the identification of molecules that inhibit, in atarget-dependent fashion, an activity of to bacterial RNAP, but that donot inhibit, or that inhibit substantially less well, an activity ofeukaryotic RNAP. This differentiation is important, because it permitsthe identification of bacterial-RNAP-selective binding molecules andbacteria-selective inhibitors.

The invention provides, by way of example only, a target regioncorresponding to, and alignable with residues 736-747 and 779-781 of theβ′ subunit of RNAP from Escherichia coli; as well as correspondingresidues of the β′ subunit of Bacillus subtilis, Haemophilus influenzae,Vibrio cholerae, Pseudomonas aeruginosa, Treponema pallidum, Borreliaburgdorferi, Xyella fastidiosa, Campylobacter jejuni, Neisseriameningitidis, Rickettsia prowazekii, Thermotoga maritima, Chlamydiatrachomatis, Mycoplasma pneumoniae, Staphylococcus aureus, Mycobacteriumtuberculosis, Synechocystis sp., Aquifex aeolicus, Deinococcusradiodurans, Thermus thermophilus, and Thermus aquaticus.

The present invention further relates to a method for identifyingmolecules that bind to the β′ pocket through the use of an assay formolecules that bind to RNAP in a β′-pocket-specific fashion. In oneembodiment, Escherichia coli RNAP or a fragment thereof containing theβ′ pocket, is used as the test protein for binding, and a derivative ofsaid RNAP or RNAP fragment having at least one a substitution, aninsertion, or a deletion within the β′ pocket is used as the controlprotein for target-site specificity of binding. “Hits” can be analyzedfor binding and inhibition of Gram-negative-bacterial RNAP,Gram-positive-bacterial RNAP, and eukaryotic RNAP I, RNAP III and RNAPIII, in vivo and in vitro. “Hits” also can be characterized structurallyby x-ray diffraction analysis of co-crystals with RNAP or an RNAPfragment containing the β′ pocket.

The invention also provides strategies to identify small-moleculeinhibitors from compound libraries. By way of example, two strategiesare described as follows: (a) selection of molecules that bind to RNAP,or a fragment thereof, in a β′-pocket-dependent fashion (affinityselection of phage-displayed linear and cyclic decapeptide libraries),and (b) screening for molecules that inhibit transcription in aβ′-pocket-dependent fashion (iterative deconvolution of solution-phaselinear and cyclic D-hexapeptide libraries). In each case, the inventionprovides the use of a wild-type bacterial RNAP, or fragment thereof, asthe test protein for binding/inhibition, and a derivative of bacterialRNAP, or a fragment thereof, having at least one substitution,insertion, or deletion within the β′ pocket as the control protein forβ′-pocket dependence of binding/inhibition.

The invention also provides for a method of identifying a compound foruse as an inhibitor of bacterial RNAP comprising: analyzing a compoundor a compound library, that involves docking to, modeling of, geometriccalculations with, and/or energetic calculations with, a portion of thestructure of an RNAP from a bacterial species comprising at least oneresidue within the set of residues corresponding to, and alignable with,the target.

The invention provides for at least three drug-discovery assay methods:a) screening based on binding of a compound within the secondary channelof a bacterial RNAP or fragment thereof; b) screening based oninhibition of an activity associated with the secondary channel of abacterial RNAP or fragment thereof; and c) screening based ondisplacement of a compound, containing a detectable group, from thesecondary channel of a bacterial RNAP or a fragment thereof.

One of the embodiments of the present invention is an assay systemdesigned to identify compounds that bind a bacterial RNAP, or a fragmentthereof, in a manner that requires the β′ pocket. The assay is designedto measure the binding of a compound to a determinant that includes atleast one amino acid residue contained within a set of amino acidresidues identifiable by sequence alignment and/or structure alignmentas corresponding to amino acid residues 736-747 and 779-781 ofEscherichia coli RNAP β′ or to amino acid residues 740-751 and 783-785of Bacillius subtilis RNAP β′.

One of the embodiments of the present invention is an assay systemdesigned to identify compounds that inhibit an activity of a bacterialRNAP, or a fragment thereof, in a manner that requires the β′ pocket.The assay is designed to measure the inhibition of an activity, saidinhibition involving the binding of a compound to a determinant thatincludes at least one amino acid residue contained within a set of aminoacid residues identifiable by sequence alignment and/or structurealignment as corresponding to amino acid residues 736-747 and 779-781 ofEscherichia coli RNAP β′ or to amino acid residues 740-751 and 783-785of Bacillius subtilis RNAP β′.

Another embodiment of the present invention is an assay designed tomeasure the binding of a compound to a bacterial RNAP derivative, or afragment thereof, containing at least one amino acid substitution,insertion, or deletion within a set of amino acid residues identifiableby sequence alignment and/or structure alignment as corresponding toamino acid residues 736-747 and 779-781 of Escherichia coli RNAP β′ orto amino acid residues 740-751 and 783-785 of Bacillius subtilis RNAPβ′.

Another embodiment of the present invention is an assay designed tomeasure the inhibition of an activity of a bacterial RNAP derivative, ora fragment thereof, containing at least one amino acid substitution,insertion, or deletion within a set of amino acid residues identifiableby sequence alignment and/or structure alignment as corresponding toamino acid residues 736-747 and 779-781 of Escherichia coli RNAP β′ orto amino acid residues 740-751 and 783-785 of Bacillius subtilis RNAPβ′.

Isolation of RNAP:

The bacterial RNAP, or RNAP derivative, can be isolated from bacteria,produced by recombinant methods, or produced through in vitro proteinsynthesis. Various compounds can be introduced to determine whether atested compound binds to, inhibits an activity of, or displaces adetectable-group containing molecule from, the bacterial RNAP or RNAPderivative in a β′-pocket-dependent manner.

Tested compounds can include antibodies, peptides, and various chemicalcompounds. Additionally, with the known amino acid sequence for aparticular RNAP, one of skill in the art could design specificinhibitors. The tested compounds can be chosen from chemical libraries,or a computer model can be used to choose compounds that are likely tobe effective based on the known structure of the secondary channel, theβ′ pocket, and the structure of the compound.

The assay system can be performed in vivo or in vitro and thus does notnecessarily require isolation of the RNAP.

The compounds can also be tested for competitive inhibition. Preferredstrategies for identifying inhibitors include: 1) through affinityselection of phage-displayed linear and cyclic decapeptide libraries and2) through iterative deconvolution of solution-phase linear and cyclicD-hexapeptide libraries. Wild type E. coli RNAP is the preferred testprotein for binding and inhibition and [Val744; Gln746]β′-RNAP (aderivative of E. coli RNAP having substitutions at two positions in theβ′(738-747) pocket) as the control protein. Deconvolution essentiallyentails the resynthesis of that combinatorial pool or mixture that wasfound to be active in screening against a target of interest.Resynthesis may result in the generation of a set of smaller pools ormixtures, or a set of individual compounds. Rescreening and iterativedeconvolution are performed until the individual compounds that areresponsible for the activity observed in the screens of the parentmixtures are isolated.

Phage Display-general Method: Searching for Peptide Ligands with anEpitope Library:

Tens of millions of short peptides can be easily surveyed for tightbinding to an antibody, receptor or other binding protein using an“epitope library.” (See (1990) Science 249:386; (1990) Science 249:404;and (1990) Proc. Natl. Acad. Sci. 87:6378). The library is a vastmixture of filamentous phage clones, each displaying one peptidesequence on the virion surface. The survey is accomplished by using thebinding protein to affinity-purify phage that display tight-bindingpeptides and propagating the purified phage in Escherichia coli. Theamino acid sequences of the peptides displayed on the phage are thendetermined by sequencing the corresponding coding region in the viralDMA's. Potential applications of the epitope library includeinvestigation of the specificity of antibodies and discovery of mimeticdrug candidates.

“Fusion phage” are filamentous bacteriophage vectors in which foreignantigenic determinants are cloned into phage gene III and displayed aspart of the gene III protein (pIII) at one tip of the virion. Fusionphage whose displayed determinant binds an antibody (Ab) can be selectedfrom a vast background of nonbinding phage by affinity purification (AP)as follows: First, phage are reacted with biotinylated Ab (bio-Ab), thendiluted and placed on a streptavidin-coated petri dish, therebyspecifically attaching Ab-reactive phage to the plastic surface throughthe Ab-biotin-streptavidin bridge. Free phage are washed away, and boundphage eluted in acid and used to infect Escherichia coli cells. A singleround of AP can enrich Ab-binding phage by as much as a factor of 10⁵relative to unreactive phage; further enrichment is achieved by furtherrounds of AP after amplification on agar medium. Thus, Ab serves as apowerful selective agent favoring the target clones, so that vastnumbers of phage can be surveyed.

The idea of using fusion phage to develop an “epitope library” (Parmleyand G. P. Smith, (1988) Gene 73:305) was inspired by the synthetic“mimotope” strategy of Geysen et al. (See Synthetic Peptides asAntigens; Ciba Foundation Symposium 119, R. Porter and J. Wheelan, Eds.(Wiley, new York. 1986), pp. 131-149). By synthesizing peptide mixtureson plastic pins and assessing their ability to bind an Ab against aprotein antigen, these workers delineated a peptide that mimics adiscontinuous epitope—an Ab-binding determinant composed of residuesdistant in the primary sequence but adjacent in the folded structure.They called these peptide mimics mimotopes. In this way ligands can bediscovered for an Ab whose specificity is not known in advance.

Fusion phage displaying short cloned peptides are infectious analogs ofchemically synthesized mimotopes, with the key advantages ofreplicability and clonability. A large library of such phage—an “epitopelibrary”—may display tens of millions of peptide epitopes. The peptidescan in effect be individually surveyed for binding to an Ab or otherbinding protein by affinity purifying reactive phage from the library,propagating individual phage clones, and sequencing the relevant part oftheir DNA's to determine the amino acid sequences of their displayedpeptides. A survey based on the epitope library undoubtedly would beimperfect because of bias introduced by the biology of the phage andother factors; still, it would represent a powerful new approach to thestudy of the specificity of Ab's and other binding proteins. (See Scottand Smith (1990) Science 249:386; Devlin et al., (1990) Science 249:404;Ciwirla et al., (1990) Proc. Natl Acad. Sci. 87:6378; McLafferty et al.,(1993) Gene 128:29; Alessandra et al., (1993) Gene 128:51; McConnell etal., (1994) Gene 151:115, which are incorporated herein by reference).

Iterative Deconvolution Approach:

See the following reference for a general discussion of iterativedeconvolution: (Ostresh et al., (1996) Meths. Enzym. 267:220, which isincorporated herein by reference). The practical development ofsynthetic combinatorial libraries (SCLs) made up of tens of millions ofcompounds has proven to be a powerful source for the identification ofnovel biologically active compounds such as analgesics, antibacterials,antifungals, and enzyme inhibitors. (See Pinilla et al., (1994) DrugDev. Res. 33:133; Pinilla et al., (1995) Pept. Sci. 37:221; Gallop etal., (1994) J. Med. Chem. 37:1233). In particular, a range of newcompounds having potent antimicrobial and/or antifungal activities havebeen rapidly identified from pools of millions of compounds. (SeeBlondelle et al., (1995) J. Appl. Bacteriol. 78-39; Blondelle et al.,(1994) Antimicrob. Agent Chemother. 38:2280; Ostresh et al., (1994)Proc. Natl. Acad. Sci. U.S.A. 31:11138; Houghten et al., (1992) BioTechniques 13:412; Houghten et al., (1991) Nature 354:84).

Nonsupport-bound SCLs, originally composed of millions of peptides, wereshown to be usable in virtually any assay system (including thoseinvolving membrane-bound acceptors or whole cell organisms). In anexpansion of SCL concepts and diversities, the original peptide SCLshave been transformed (i.e., peralkylated and/or exhaustively reduced)using a “libraries from libraries” approach (Ostresh et al., (1994)Proc. Nail. Acad. Sci. U.S.A. 91:11138; Dörner et al., in “Peptides1994: Proceedings of the 23rd European Peptide Symposium” (H. L. S.Maia, ed.), p. 463. Escom, Leiden, 1995; and Cuervo et al., Id. at page465) to yield peptidomimetic and organic libraries having entirelydifferent physical, chemical, and biological properties relative to thepeptide SCLs used as starting materials. The screening of such librarieshas yielded active compounds derived from entirely different sequencesthan the active peptides previously identified from the starting SCLsusing the same assay.

Two approaches can be employed for the structural deconvolution ofactive compounds from assay data using nonsupport-bound SCLs: the“iterative” approach and the “positional scanning” approach. Inaddition, two synthetic methods were developed for the incorporation ofmultiple functionalities at diverse positions within an SCL. As firstillustrated for peptides, (See Houghten et al., (1992) Bio Techniques13:412; and Houghten et al., (1991) Nature 354:84, which areincorporated herein by reference). The first synthetic method, known asthe “divide, couple, and recombine” (DCR) (Id.) or “split resin” (Lam etal., (1991) Nature 354:82) method, has typically been used with theiterative deconvolution approach. The second synthetic method, whichinvolves the use of a predefined chemical ratio of protected amino acidsat each coupling step for incorporation of mixture positions, Ostresh etal., (1994) Biopolymers 34:1681) has been developed for use with thepositional scanning deconvolution process (Pinilla et al., (1992)BioTechniques 13:901). This latter method offers the advantage that bothdefined and mixture positions are easily incorporated at any position ina sequence.

These synthesis and deconvolution methods have been used to identifyindividual active compounds in a wide variety of SCLs and assays.(Pinilla et al., (1994) Drug Dev. Res. 33:133; Pinilla et al., (1995)Pept. Sci. 37:221). More specifically, individual compounds fromnonsupport-bound SCLs have been identified which have potentantimicrobial activity against gram-positive bacteria (Staphylococcusaureus, Streptococcus sanguis), gram-negative bacteria (Escherichiacoli, Pseudomonas aeruginosa), and fungi (Candida albicans). Theiterative deconvolution approach will be illustrated here for thepreparation of a dual-defined position hexapeptide SCL, designatedOOXXXX—NH₂ (where O represents a defined amino acid, and X represents amixture of amino acids) using the DCR method. The mixtures making upthis library have been assayed for antimicrobial and/or antifungalactivity (Blondelle et al., (1995) Trends Anal. Chem. 14:83; Houghten etal., (1992) Bio Techniques 13:412; and Houghten et al., (1991) Nature354:84) in order to identify the first two amino acid residues of activehexapeptide sequences. The remaining four positions were then identifiedsequentially through an iterative process of synthesis and screening.This process can be completed in 6 to 10 weeks (four separate iterativesynthesis steps are required). The positional scanning approach,involves the screening of separate single position SCLs to identify themost, effective amino acids at each position of the sequence. When usedin concert, this information can be used to identify individual activesequences. This process can be completed in approximately 2 weeks (onlyone synthesis step is required for confirmation of activity).

Both iterative and positional scanning peptide SCLs have been used asstarting materials for the generation of peptidomiraetic SCLs using the“libraries from libraries” approach.

Synthesis:

Iterative Peptide Libraries; As mentioned earlier, these libraries areprepared using the DCR process (Houghten et al., (1991) Nature 354:84)(in conjunction with simultaneous multiple peptide synthesis (SMPS),(Houghten, (1985) Proc. Natl. Acad. Sci. U.S.A. 82:5131) also known asthe “tea bag” approach. Standard t-butyloxycarbonyl (Boc)-based peptidesynthesis protocols are typically used to couple protected amino acids(Bachem, Torrance, Calif.) to methylbenzhydrylamine (MBHA)-derivatizedpolystyrene resin (Peninsula, Belmont, Calif.).Fluorenylmethyloxycarbonyl (Emoc)-based chemistry strategies can also beused. During preparation of the initial library, a portion of each resinmixture (i.e., X-resin, XX-resin, XXX-resin, etc) is held back forsynthesis of the subsequent peptide mixtures during the iterativeprocess in which additional positions are sequentially defined. While upto 76 amino acids have been used in the mixture positions, cysteine isnormally omitted from the mixture positions of an SCL to preventpolymerisation side reactions. It should be noted that for librariessynthesized by the DCR method, the number of resin beads used should be10 to 100 times higher than the final number of individual compounds ina resin mixture in order to ensure statistical representation of eachpeptide in the library (Gallop et al., (1994) I. Med. Chem. 37:1233).The generation of a dual-defined position SCL made up of L-amino acidhexapeptides (designated OOXXXX—NH₂) is described here to illustrate theOCR methodology. This library contains approximately 52 million(20²×19⁴) different peptides.

The practical use of nonsupport-bound combinatorial libraries representsan important breakthrough in all areas of basic research and drugdiscovery. The use of a wide variety of chemical transformations permitsa range of peptidomimetic libraries to be generated, which greatlyexpands the chemical diversity available. The results described in thischapter demonstrate that an existing peptide PS-SCL can be chemicallytransformed to generate a peptidomimetic SCL from which highly activeindividual compounds can be identified. The synthesis and deconvolutionmethods developed for peptide libraries are easily applied to othertypes of chemical pharmacophores. The soluble nature of thenonsupport-bound combinatorial libraries is a distinct advantage overother methods in that membrane-bound and whole cell assays can also beused. In addition, the deconvolution methods used allow the chemicalstructure of peptidic, peptidomimetic, and organic compounds to bedetermined based solely on the structural similarities of compoundswithin each active pool or sublibrary.

Screening for an Inhibitor of Bacterial RNAP:

One aspect of the invention provides high throughput screening ofmolecules specific to the bacterial RNAP target. This can be done inmany different ways well known in the art. For example, this could bedone by attaching bacterial RNAP to the bottom of the wells of a 96-wellplate at an appropriate concentration by incubating the RNAP in the wellovernight at 4° C. Alternatively, the wells are first coated withcompositions of polylysine that facilitates the binding of the bacterialRNAP to the wells. Following attachment, samples from a library of testcompounds (concentrations are determined by the compound being tested)are added (along with an appropriate binding buffer known in the art) tothe wells and incubated for a sufficient time and temperature tofacilitate binding. Following the incubation, the wells are washed withan appropriate washing solution at 4° C. Increasing or decreasing saltand/or detergent concentrations in the wash varies the stringency of thewashing steps. Detection of binding is accomplished using antibodies(representative examples include RIA and ELISA), biotinylation,biotin-streptavidin binding, and radioisotopes. The concentration of thesample library compounds is also varied to calculate a binding affinityby Scatchard analysis. Binding to the bacterial RNAP target identifies a“lead compound”. Once a lead compound is identified, the screeningprocess is repeated using compounds chemically related to the leadcompound to identify compounds with the tightest binding affinities.Selected compounds having binding affinity are further tested in one oftwo assays. These assays use test compounds from 1) phage-displayedlinear and cyclic decapeptide libraries and 2) iterative deconvolutionof solution-phase linear and cyclic D-hexapeptide libraries.

A phage library can be used to test compounds that could bind to the β′pocket of bacterial RNAP. The phage library is constructed in theN-terminal region of the major coat protein pVIII, as previouslydescribed (Felici et al., 1991). In addition, in an attempt to define amore constrained context, two Cys are included as invariant residuesflanking the random nonapeptide. Transformation yields approximately1×10⁸ independent clones, and the presence of a productive insert isindicated by the blue color of the colonies on Xgal/IPTG plates (Feliciet al., 1991). The construction of the library results in hybridcapsids, expressing the random peptides, dispersed along wt pVIIIcopies. The absence of the Cys in wt pVIII allows one to detect thepresence of free thiol groups in the hybrid capsids. Clones are analyzedwith a Cys-specific recompound (DIG protein detection kit, BoehringerMannheim, Germany) in order to show some of the peptides are in cyclizedform. This indicates that in many cases the insert is displayed as aloop structure, which limits its mobility. Phage affinity purificationis performed utilizing the biopanning technique, as previously describedby Parmley and Smith (1988). After the round of biopanning, 10⁴ phageout of the initial 10¹⁰ are eluted from a streptavidin-coated plate. Thephage are screened directly with a plaque assay. Single plagues (10⁵)are transferred onto nitrocellulose and probed with RNAP. Positiveplaques are eluted from nitrocellulose, the phage are amplified andsequenced, and their reactivity is further confirmed by dot-blotanalysis. The amino acid sequences are then deduced.

Biologically active compounds are selected from large populations ofrandomly generated sequences. Libraries are made up of six-residuepeptide sequences with amidated carboxy-termini and either acetylated ornon-acetylated amino-termini. The first two amino acids in each peptidechain are individually and specifically defined, while the last fouramino acids consist of equimolar, or close to equimolar, mixtures of 19of the 20 naturally occurring L-amino acids, cysteine is omitted fromthe mixture positions of the two libraries but included in the definedpositions. The peptides in these libraries are generally represented asAc—O₁O₂XXXX—NH₂ and O₁O₂XXXX—NH₂, where O₁ and O₂ are defined positions,which are represented by the single letters AA, AC, AD and so on up toand including YV, YW, YY, to reach a total of 400 (20²) combinations,and each X position is represented by an equimolar mixture of the 19natural amino acids (non-natural amino acids can be used as well). Fourmixture positions (XXXX) result in a total of 130,321 (19⁴) differentcombinations. Each of the 400 different peptide mixtures that make upeach of the libraries thus consists of 130,321 individual hexamers; intotal, 52,321,400 peptides are represented. The peptides are attached toa resin or alternatively cleaved from the resin, extracted andlyophilized before use. Each nonsupport-bound peptide mixture istypically used at a concentration of 1 mg/ml. Therefore, if one assumesthat the average molecular weight of Ac—O₁O₂XXXX—NH₂ is 785, then amixture of 130,321 peptides at a total final concentration of 1 mg/mlyields a concentration of every peptide within each mixture of 7.67ng/ml (9.8 nM), sufficiently high for most biologically significantinteractions if even a single peptide sequence is active. After themixture of libraries is screened for binding to bacterial RNAP, theremaining mixture positions are defined through an iterative enhancementand selection process in order to identify the most active sequence. Anextremely rapid alternative method for identifying active compounds isthe positional scanning approach. In this approach, if one uses alibrary made up of peptides, for example, each of the individualsub-libraries (one for each position along the peptide) that make up thepositional scanning library is composed of 18 different peptidemixtures. Each position is defined (represented as O) and occupied byone of 18 of the 20 natural L-amino acids (cysteine and tryptophan areomitted); the remaining five positions of the six-residue sequence arecomposed of mixtures (represented as X) of the same 18 amino acids. Thesix different sub-libraries vary only in the location of their definedamino acids, they can therefore be represented as: Ac—O₁XXXXX—NH₂,Ac—XO₂XXXX—NH₂, Ac—XXO₃XXX—NH₂, Ac—XXXO₄XX—NH₂, AC—XXXXO₅X—NH₂, andAc—XXXXXO₆—NH₂. As each peptide mixture represents 1,889,568 (18⁵)individual sequences, each of the six positional sub-libraries containsin total 34,012,224 (18×1,883,568) different compounds. Alternatively,each of the six individual sub-libraries (for example, AC—XXXO₄XX—NH₂)can be examined independently and moved forward in an interactivefashion. When used in concert, however, this set of 108 mixturesconstitutes a positional scanning library.

Screening Assays for Compounds that Interfere with the Interaction ofRNAP Target and MccJ25:

The β′ pocket of bacterial RNAP—which contains the target—and compoundswhich interact and bind are sometimes referred to herein as “bindingpartners.” Any of a number of assay systems may be utilized to testcompounds for their ability to interfere with the interaction of thebinding partners. However, rapid, high-throughput assays for screeninglarge numbers of compounds, including but not limited to ligands(natural or synthetic), peptides, or small organic molecules arepreferred. Compounds that are so identified to interfere with theinteraction of the binding partners should be further evaluated forbinding or inhibitory activity in cell based assays, animal modelsystems and in patients as described herein. The basic principle of theassay systems used to identify the compounds of the present invention isbased on the identification of compounds that interfere with theinteraction between the target and MccJ25, which involves preparing areaction mixture containing a bacterial RNAP, or RNAP fragment orderivative containing the secondary channel, and MccJ25 under conditionsand for a time sufficient to allow the two binding partners to interactand bind, thus forming a complex. In order to test a compound forinhibitory activity, the reaction is conducted in the presence andabsence of the test compound; i.e., the test compound may be initiallyincluded in the reaction mixture, or added at a time subsequent to theaddition of the bacterial RNAP, or RNAP fragment or derivativecontaining the secondary channel, and MccJ25; controls are incubatedwithout the test compound. The formation of a complex between thebacterial RNAP secondary channel and MccJ25 then is detected. Theformation of a complex in the control reaction, but not, or to a lesserextent, in the reaction mixture containing the test compound indicatesthat the compound interferes with the interaction between the bacterialRNAP, or RNAP fragment or derivative containing the secondary channel,and MccJ25.

The assay components and various formats that may be utilized aredescribed in the subsections below.

Assay Components:

The bacterial RNAP, or RNAP fragment or derivative containing thesecondary channel, and MccJ25 binding partners used as components in theassay may be derived from natural sources, e.g., purified from bacterialRNAP, respectively, using protein separation techniques well known inthe art; produced by recombinant DNA technology using techniques knownin the art (see e.g., Sambrook et al., 1989, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratories Press, Cold SpringHarbor, N.Y.); and/or chemically synthesized in whole or in part usingtechniques known in the art; e.g., peptides can be synthesized by solidphase techniques, cleaved from the resin and purified by preparativehigh performance liquid chromatography (see, e.g., Creighton, 1983,Proteins: Structures and Molecular Principles, W. H. Freeman & Co.,N.Y., pp. 50-60). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing; e.g., using the Edmandegradation procedure (see e.g., Creighton, 1983, supra at pp. 34-49).

One of the binding partners used in the assay system should be labeled,either directly or indirectly, to facilitate detection of a complexformed between the bacterial RNAP secondary channel and MccJ25. Any of avariety of suitable labeling systems may be used including but notlimited to radioisotopes such as ¹²⁵I and ³²P; enzyme labelling systemsthat generate a detectable colorimetric signal or light when exposed tosubstrate; and fluorescent labels.

Fluorescent labels are preferred. Cyanine fluorescent labels areespecially preferred. Cyanine labels incorporated at residue 13 or 15 ofMccJ25 are especially preferred.

Where recombinant DNA technology is used to produce the bacterial RNAP,or RNAP fragment or derivative containing the secondary channel, andMccJ25 binding partners of the assay it may be advantageous to engineerfusion proteins that can facilitate labeling, immobilization and/ordetection. For example, the coding sequence of the bacterial RNAPsecondary channel can be fused to that of a heterologous protein thathas enzyme activity or serves as an enzyme substrate in order tofacilitate labeling and detection. The fusion constructs should bedesigned so that the heterologous component of the fusion product doesnot interfere with binding of the bacterial RNAP secondary channel andMccJ25.

Indirect labeling involves the use of a third protein, such as a labeledantibody, which specifically binds to the bacterial RNAP secondarychannel. Such antibodies include but are not limited to polyclonal,monoclonal, chimeric, single chain, Fab fragments and fragments producedby an Fab expression library.

For the production of antibodies, various host animals may be immunizedby injection with the bacterial RNAP secondary channel. Such hostanimals may include but are not limited to rabbits, mice, and rats, toname but a few. Various adjuvants may be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

Monoclonal antibodies may be prepared by using any technique, whichprovides for the production of antibody molecules by continuous celllines in culture. These include but are not limited to the hybridomatechnique originally described by Kohler and Milstein, (Nature, 1975,256:495-497), the human B-cell hybridoma technique (Kosbor et al., 1983,Immunology Today, 4:72, Cote et al., 1983, Proc. Natl. Acad. Sci.,80:2026-2030) and the EBV-hybridoma technique (Cole et al., 1985,Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96). In addition, techniques developed for the production of“chimeric antibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci.,81: 6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda etal., 1985, Nature, 314:452-454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused. Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,946,778) can be adapted to producesingle chain antibodies specific to the bacterial RNAP secondarychannel.

Antibody fragments, that recognize specific epitopes may be generated byknown techniques. For example, such fragments include but are notlimited to: the F(ab′)₂ fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragments.Alternatively, Fab expression libraries may be constructed (Huse et al.,1989, Science, 246:1275-1281) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

Assay Formats:

The assay can be conducted in a heterogeneous or homogeneous format. Aheterogeneous assay is an assay in which reaction results are monitoredby separating and detecting at least one component during or followingreaction. A homogeneous reaction is an assay in which reaction resultsare monitored without separation of components. In either approach, theorder of addition of reactants can be varied to obtain differentinformation about the compounds being tested. For example, testcompounds that interfere with the interaction between the bindingpartners, e.g., by competition, can be identified by conducting thereaction in the presence of the test substance; i.e., by adding the testsubstance to the reaction mixture prior to or simultaneously with thebacterial RNAP secondary channel and MccJ25. On the other hand, testcompounds that disrupt preformed complexes, e.g. compounds with higherbinding constants that displace one of the binding partners from thecomplex, can be tested, by adding the test compound to the reactionmixture after complexes have been formed. The various formats aredescribed briefly below.

In one example of a heterogeneous assay system, one binding partner,e.g., either the bacterial RNAP secondary channel or MccJ25, is anchoredonto a solid surface, and its binding partner, which is not anchored, islabeled, either directly or indirectly. In practice, microtiter platesare conveniently utilized. The anchored species may be immobilized bynon-covalent or covalent attachments. Alternatively, an immobilizedantibody specific for the bacterial RNAP secondary channel may be usedto anchor the bacterial RNAP secondary channel to the solid surface. Thesurfaces may be prepared in advance and stored.

In order to conduct the assay, the binding partner of the immobilizedspecies is added to the coated surface with or without the testcompound. After the reaction is complete, unreacted components areremoved (e.g., by washing) and any complexes formed will remainimmobilized on the solid surface. The detection of complexes anchored onthe solid surface can be accomplished in a number of ways. Where thebinding partner was pre-labeled, the detection of label immobilized onthe surface indicates that complexes were formed. Where the bindingpartner is not pre-labeled, an indirect label can be used to detectcomplexes anchored on the surface; e.g., using a labeled antibodyspecific for the binding partner (the antibody, in turn, may be directlylabeled or indirectly labeled with a labeled anti-Ig antibody).Depending upon the order of addition of reaction components, testcompounds which inhibit complex formation or which disrupt preformedcomplexes can be detected.

Alternatively, the reaction can be conducted in a liquid phase in thepresence or absence of the test compound, the reaction productsseparated from unreacted components, and complexes detected; e.g., usingan immobilized antibody specific for an epitope on the bacterial RNAPsecondary channel to anchor any complexes formed in solution. Again,depending upon the order of addition of reactants to the liquid phase,test compounds which inhibit complex or which disrupt preformedcomplexes can be identified.

In a preferred embodiment of the invention, a homogeneous assay can beused. In this approach, a preformed complex of the bacterial RNAPsecondary channel and MccJ25 is prepared in which one of the bindingpartners is labeled, but the signal generated by the label is quencheddue to complex formation (see, e.g., U.S. Pat. No. 4,109,496 byRubenstein, which utilizes this approach for immunoassays). The additionof a test substance that competes with and displaces one of the bindingpartners from the preformed complex will result in the generation of asignal above background. In this way, test substances, which disrupt thebacterial RNAP secondary channel and MccJ25 interaction can beidentified.

One aspect of the invention provides for developing methods to usefluorescence resonance energy transfer (FRET)-based homogeneous assaysto provide probe-labelled derivatives of the MccJ25. (Förster, 1948;reviewed in Lilley and Wilson. 2000; Selvin, 2000; Mukhopadhyay et al.,2001; Mekler et al., 2002). FRET occurs in a system having a fluorescentprobe serving as a donor and a second fluorescent probe serving as anacceptor, where the emission wavelength of the donor overlaps theexcitation wavelength of the acceptor. In such a system, upon excitationof the donor with light of its excitation wavelength, energy can betransferred from the donor to the acceptor, resulting in excitation ofthe acceptor and omission at the acceptor's emission wavelength.

With commonly used fluorescent probes, FRET permits accuratedetermination of distances in the range of ˜20 to ˜100 Å. FRET permitsaccurate determination of distances up to more than one-half thediameter of a transcription complex (diameter ˜150 Å; Zhang et al. 1999;Cramer et al., 2001; Gnatt et al., 2001).

A preferred assay involves monitoring of FRET between afluorescent-probe-labeled derivative of a bacterial RNAP and afluorescent-probe-labeled derivative of MccJ25.

An especially preferred assay involves monitoring of FRET between acoumarin-dye-labeled derivative of a bacterial RNAP and acyanine-dye-labeled derivative of MccJ25. Especially preferred sites oflabeling of RNAP for this purpose include residue 14, residue 59, orresidue 517 of Escherichia coli RNAP σ⁷⁰ subunit, or a correspondingresidue, identifiable by sequence and/or structural alignment, ofanother bacterial RNAP σ subunit. Especially preferred sites of labelingof MccJ25 for this purpose include residue 13 and residue 15.

In accordance with the invention, a given compound found to inhibit onebacterium may be tested for general antibacterial activity against awide range of different bacterial species. For example, and not by wayof limitation, a compound that inhibits the interaction of Escherichiacoli RNAP, or a RNAP fragment or derivative thereof containing thesecondary channel, of with MccJ25 can be tested, according to the assaysdescribed infra, against Haemophilus influenzae.

Assays for Antibacterial Activity:

Any of the inhibitory compounds, which are identified in the foregoingassay systems, may be tested for antibacterial activity in any one ofthe various microbiological assays known to the skilled worker in thefield of microbiology.

Animal Model Assays:

The most effective inhibitors of bacterial RNAP identified by theprocesses of the present invention can then be used for subsequentanimal experiments. The ability of an inhibitor to prevent bacterialinfection can be assayed in animal models that are natural hosts forbacteria. Such animals may include mammals such as pigs, dogs, ferrets,mice, monkeys, horses, and primates. As described in detail herein, suchanimal models can be used to determine the LD₅₀ and the LD₅₀ in animalsubjects, and such data can be used to derive the therapeutic index forthe inhibitor of the bacterial RNAP secondary channel/MccJ25interaction.

Pharmaceutical Preparations and Methods of Administration:

The identified compounds that inhibit bacterial replication can beadministered to a patient at therapeutically effective doses to treatbacterial infection. A therapeutically effective dose refers to thatamount of the compound sufficient to result in amelioration of symptomsof bacterial infection.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of infection in order to minimize damage to uninfected cells andreduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal infection, or ahalf-maximal inhibition) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers or excipients.

Thus, the compounds and their physiologically acceptable salts andsolvates may be formulated for administration by inhalation orinsufflation (either through the mouth or the nose) or oral, buccal,parenteral or rectal administration.

For administration by inhalation, the compounds fox use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebuliser, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycollate); or wetting agents (e.g., sodium lauryl sulphate).The tablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

As used herein a “small molecule” is a compound that has a molecularweight of less than 15 kDa.

As used herein a “small organic molecule” is an organic compound [ororganic compound complexed with an inorganic compound (e.g., metal)]that has a molecular weight of less than 3 kDa.

As used herein the term “about” means within 10 to 15%, preferablywithin 5 to 10%. For example an amino acid sequence that contains about60 amino acid residues can contain between 51 to 69 amino acid residues,more preferably 57 to 63 amino acid residues.

As used herein the term “target” minimally comprises amino acid residuesof a target set of residues corresponding to, and alignable with, eitherresidues 736-747 and 779-781 of the β′ subunit of RNAP from Escherichiacoli or a set of residues corresponding to, and alignable with residues740-751 and 783-785 of the β′ subunit of RNAP from Bacillus subtilis.

As used herein, the term “sequence homology” in all its grammaticalforms refers to the relationship between proteins that possess a “commonevolutionary origin,” including proteins from superfamilies (e.g., theimmunoglobulin superfamily) and homologous proteins from differentspecies (e.g., myosin light chain, etc). [Reeck et al., Cell, 50:667(1987)].

Accordingly, the term “sequence similarity” in all its grammatical formsrefers to the degree of identity or correspondence between nucleic acidor amino acid sequences of proteins that do not share a commonevolutionary origin [see Reeck et al., 1987, supra]. However, in commonusage and in the instant application, the term “homologous,” whenmodified with an adverb such as “highly,” may refer to sequencesimilarity and not a common evolutionary origin.

Similarly, in a particular embodiment, two amino acid sequences are“substantially homologous” or “substantially similar” when greater than25% of the amino acids are identical, or greater than about 50% aresimilar (functionally identical). Preferably, the similar or homologoussequences are identified by alignment using, for example, the GCG(Genetics Computer Group, Program Manual for the GCG Package, Version 7,Madison, Wis.) pileup program with the default parameters.

The term “corresponding to” is used herein to refer similar orhomologous sequences, whether the exact position is identical ordifferent from the molecule to which the similarity or homology ismeasured. Thus, the term “corresponding to” refers to the sequencesimilarity, and not the numbering of the amino acid residues ornucleotide bases.

The present invention contemplates isolation of nucleic acids encodingeither targets I or I. The present invention further provides forsubsequent modification of the nucleic acid to generate a fragment ormodification of the target, that will crystallize.

Protein-structure Based Design of Inhibitors of Bacterial RNAP:

Once the three-dimensional structure of a crystal comprising a bacterialRNAP target is determined, a potential modulator of the target, can beexamined through the use of computer modeling using a docking programsuch as GRAM, DOCK, or AUTODOCK [Dunbrack et al., Folding & Design,2:27-42 (1997)], to identify potential modulators of the bacterial RNAPtarget. This procedure can include computer fitting of potentialmodulators to the bacterial RNAP target to ascertain how well the shapeand the chemical structure of the potential modulator will bind toeither the individual bound subunits or to the bacterial RNAP target[Bugg et al., Scientific American, December:92-98 (1993); West et al.,TIPS, 16:67-74 (1995)]. Computer programs can also be employed toestimate the attraction, repulsion, and steric hindrance of the subunitswith a modulator/inhibitor (e.g., the bacterial RNAP target and apotential stabilizer).

Initially, compounds known to bind to the target—for example, MccJ25—canbe systematically modified by computer modeling programs until one ormore promising potential analogs are identified. In addition, systematicmodification of selected analogs can then be systematically modified bycomputer modeling programs until one or more potential analogs areidentified. Such analysis has been shown to be effective in thedevelopment of HIV protease inhibitors [Lam et al., Science 263:330-384(1994); Wlodawer et al., Ann. Rev. Biochem. 62:543-585 (1993); Appelt,Perspectives in Drug Discovery and Design 1:23-48 (1993); Erickson,Perspectives in Drug Discovery and Design 1:109-128 (1993)].Alternatively, a potential modulator could be obtained by initiallyscreening a random peptide library produced by recombinant bacteriophagefor example, [Scott and Smith, Science, 249:386-390 (1990); Cwirla etal., Proc. Natl. Acad. Sci., 87:6378-6382 (1990); Devlin et al.,Science, 249:404-406 (1990)]. A peptide selected in this manner wouldthen be systematically modified by computer modeling programs asdescribed above, and then treated analogously to a structural analog asdescribed below.

Once a potential modulator/inhibitor is identified, it can be eitherselected from a library of chemicals as are commercially available frommost large chemical companies including Merck, Glaxo Welcome, BristolMeyers Squib, Monsanto/Searle, Eli Lilly, Novartis and Pharmacia UpJohn,or alternatively the potential modulator may be synthesized de novo. Asmentioned above, the de novo synthesis of one, or even a group of,specific compounds is reasonable in the art of drug design. Thepotential modulator can be placed into a standard binding assay withRNAP or an active fragment thereof such as the target, for example. Thesubunit fragments can be synthesized by either standard peptidesynthesis described above, or generated through recombinant DNAtechnology or classical proteolysis. Alternatively, the correspondingfull-length proteins may be used in these assays.

For example, the β′ subunit can be attached to a solid support. Methodsfor placing the β′ subunit on the solid support are well known in theart and include such things as linking biotin to the β′ subunit andlinking avidin to the solid support. The solid support can be washed toremove unreacted species. A solution of a labeled potential modulator(e.g., an inhibitor) can be contacted with the solid support. The solidsupport is washed again to remove the potential modulator not bound tothe support. The amount of labeled potential modulator remaining withthe solid support and thereby bound to the β′ subunit can be determined.Alternatively, or in addition, the dissociation constant between thelabeled potential modulator and the β′ subunit, for example can bedetermined. Suitable labels for bacterial RNAP target or the potentialmodulator are exemplified herein. In a particular embodiment, isothermalcalorimetry can be used to determine the stability of the bacterial RNAPtarget in the absence and presence of the potential modulator.

In another aspect of the present invention, a compound is assayed forits ability to bind to the target. A compound that binds to the targetthen can be selected. In a particular embodiment, the effect of apotential modulator on an activity of the bacterial RNAP target isdetermined. The potential modulator then can be added to a bacterialculture to ascertain its effect on bacterial proliferation. A potentialmodulator that inhibits bacterial proliferation then can be selected.

In a particular embodiment, the effect of the potential modulator on anactivity of a bacterial RNAP, or a fragment thereof, is determined(either independently, or subsequent to a binding assay as exemplifiedabove). In one such embodiment, the extent or rate of the DNA-dependentRNA transcription is determined. For such assays, a labeled nucleotidecould be used. This assay can be performed using a real-time assay—e.g.,with a fluorescent analog of a nucleotide. Alternatively, thedetermination can include the withdrawal of aliquots from the incubationmixture at defined intervals and subsequent placing of the aliquots onnitrocellulose paper or on gels. In a particular embodiment thepotential modulator is selected when it is an inhibitor of the bacterialRNAP.

One assay for RNAP activity is a modification of the method of Burgesset al. [J. Biol. Chem., 244:6160 (1969)]. One unit incorporates onenanomole of UMP into acid insoluble products in 10 minutes at 37° C.under the assay conditions such as those listed below. The suggestedrecompounds are: (a) 0.04 M Tris-HCl, pH 7.9, containing 0.01 M MgCl₂,0.15 M KCl, and 0.5 mg/ml BSA; (b) Nucleoside triphosphates (NTP): 0.15mM each of ATP, CTP, GTP, UTP; spiked with ³H-UTP 75000-150000 25cpms/0.1 ml; (c) 0.15 mg/ml calf thymus DNA; (d) 10% cold perchloricacid; and (e) 1% cold perchloric acid. 0.1-0.5 units of RNAP in 5 μl-10μl is used as the starting enzyme concentration.

The procedure is to add 0.1 ml Tris-HCl, 0.1 ml NTP and 0.1 ml DNA to atest tube for each sample or blank. At zero time enzyme (or buffer forblank) is added to each test tube, and the contents are then mixed andincubated at 37° C. for 10 minutes. 1 ml of 10% perchloric acid is addedto the tubes to stop the reaction. The acid insoluble products can becollected by vacuum filtration through MILLIPORE filter discs having apore size of 0.45 u-10 u (or equivalent). The filters are then washedfour times with 1% cold perchloric acid using 1 ml-3 ml for each wash.These filters are then placed in scintillation vials. 2 mls of methylcellosolve are added to the scintillation vials to dissolve the filters.When the filters are completely dissolved (after about five minutes) 10mls of scintillation fluid are added and the vials are counted in ascintillation counter.

Additional assays for analysis of RNAP activity contemplated by thepresent invention include RNA transcription assays andfluorescence-detected abortive initiation assays described in detailbelow, concerning defining the target of MccJ25. The present inventionfurther provides for assays for analysis of antibacterial activity, suchas for example include a Minimal Bacteriocidal Concentration (MBC) assayalso described in detail below, concerning defining the target ofMccJ25.

For calculation of units of RNAP/mg of protein the equation described inU.S. Pat. No. 6,225,076 can be used.

When suitable potential modulators are identified, a crystal can begrown that comprises the bacterial RNAP, or a fragment thereof, and thepotential modulator. Preferably, the crystal effectively diffractsX-rays for the determination of the atomic coordinates of theprotein-ligand complex to a resolution of better than 4.0 Angstroms. Thethree-dimensional structure of the crystal is determined by molecularreplacement. Molecular replacement involves using a knownthree-dimensional structure as a search model to determine the structureof a closely related molecule or protein-ligand complex in a new crystalform. The measured X-ray diffraction properties of the new crystal arecompared with the search model structure to compute the position andorientation of the protein in the new crystal. Computer programs thatcan be used include: X-PLOR (see above), CMS, (Crystallography and NMRSystem, a next level of XPLOR), and AMORE [J. Navaza, ActaCrystallographies ASO, 157-163 (1994)]. Once the position andorientation are known an electron density map can be calculated usingthe search model to provide X-ray phases. Thereafter, the electrondensity is inspected for structural differences and the search model ismodified to conform to the new structure. Using this approach, if willbe possible to solve the three-dimensional structure of differentbacterial target having pre-ascertained amino acid sequences. Othercomputer programs that can be used to solve the structures of thebacterial RNAP from other organisms include: QUANTA, CHARMM; INSIGHT;SYBYL; MACROMODE; and ICM.

A candidate drug can be selected by performing rational drug design withthe three-dimensional structure determined for the crystal, preferablyin conjunction with computer modeling discussed above. The candidatedrug (e.g., a potential modulator of bacterial RNAP) can then be assayedas exemplified above, or in situ. A candidate drug can be identified asa drug, for example, if it inhibits bacterial proliferation.

A potential inhibitor (e.g., a candidate antibacterial agent) would beexpected to interfere with bacterial growth. Therefore, an assay thatcan measure bacterial growth may be used to identify a candidateantibacterial agent.

Methods of testing a potential bacteriostatic or bacteriocidal compound(e.g., the candidate antibacterial agent) in isolated cultures and inanimal models are well known in the art, and can include standardminimum-inhibitory-concentration (MIC) andminimum-bacteriocidal-concentration (MBC) assays. In animal models, thepotential modulators can be administered by a variety of ways includingtopically, orally, subcutaneously, or intraperitoneally depending on theproposed use. Generally, at least two groups of animals are used in theassay, with at least one group being a control group, which isadministered the administration vehicle without the potential modulator.

For all of the assays described herein further refinements to thestructure of the compound generally will be necessary and can be made bythe successive iterations of any and/or all of the steps provided by theparticular screening assay.

It is anticipated that compounds identified according to the target andmethod of this invention would have applications not only inantibacterial therapy, as described above, but also in: (i)identification of bacterial RNAP (diagnostics, environmental-monitoring,and sensors applications), (ii) labeling of bacterial RNAP (diagnostics,environmental-monitoring, imaging, and sensors applications), (iii)immobilization of bacterial RNAP (diagnostics, environmental-monitoring,and sensors applications), (iv) purification of bacterial RNA polymerase(biotechnology applications), (v) regulation of bacterial geneexpression (biotechnology applications), and (vi) antisepsis(antiseptics, disinfectants, and advanced-materials applications).

The present invention is not to be limited in scope by the specificembodiments describe herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

EXAMPLES Example 1 Cyclic Peptide MccJ25 (MccJ25) Inhibits Transcriptionby Obstructing the RNAP Secondary Channel

Introduction:

The cyclic-peptide antibiotic MccJ25 (MccJ25) inhibits transcription byEscherichia coli RNA polymerase (RNAP) in a purified system. Biochemicalresults indicate that MccJ25 inhibits the abortive-initiation andelongation phases of transcription. Kinetic results indicate that MccJ25inhibits transcription by interfering with NTP uptake. Genetic resultsindicate that MccJ25 inhibits transcription through interactions with anextensive determinant, comprising nearly fifty amino acid residues,within the RNAP secondary channel (also known as the secondary channelor pore). MccJ25 inhibits transcription by binding within, andobstructing, the RNAP secondary channel—acting essentially as a “cork ina bottle.” Obstruction of the RNAP secondary channel represents a novelmechanism of inhibition and an attractive target for drug discovery.

MccJ25 (MccJ25) is a 21 residue cyclic-peptide antibiotic (Salomon andFarias (1992) J. Bacteriol. 174, 7428-7435; Blond et al., (1999) Eur. J.Biochem. 259, 747-755). MccJ25 is produced by Escherichia coli strainsthat harbor a plasmid-borne antibiotic-synthesis and antibiotic-exportcassette, consisting of a gene for MccJ25 precursor (a 58 residue linearpeptide), two genes for factors that process MccJ25 precursor intoMccJ25, and one gene for export of MccJ25 (Solbiati et al., (1993) J.Bacteriol. 181, 2659-2662; Duarte et al., (2001) Can. J. Microbiol. 47,877-882). MccJ25 exhibits bacteriocidal activity against a range ofGram-negative bacterial species, including E. coli (Salomon and Farias(1992) J. Bacteriol. 174, 7428-7435).

Recently, it was proposed that the functional target of MccJ25 is RNAP(RNAP). Thus, it reported that it is possible to isolate a mutantresistant to MccJ25 in rpoC—the gene for the RNAP β′ subunit—and thatMccJ25 inhibits RNAP-dependent transcription in crude cell extracts(Delgado et al., (2001) J. Bacteriol. 183, 4543-4550).

The present invention provides that MccJ25 inhibits transcription in apurified system, provides the steps in transcription at which inhibitionoccurs, provides the mechanism by which inhibition occurs, and providesdefine—through isolation of more than 120 independentsingle-substitution MccJ25-resistant mutants of rpoC following randomand saturation mutagenesis—the determinant of RNAP for function ofMccJ25. The present invention also provides that MccJ25 inhibitstranscription by binding within, and obstructing, the RNAP secondarychannel. This represents a novel mechanism for inhibition of anucleotide polymerase and an attractive target for antibacterial drugdiscovery.

MccJ25 Does not Inhibit Open-Complex Formation:

Transcription involves the following steps (Record et al., (1996) InEscherichia coli and Salmonella, F. C. Neidhart, ed. (Washington, D.C.,ASM Press), pp. 792-820; DeHaseth et al., (1998) J. Bacteriol. 180,3019-3025): (i) RNAP binds to promoter DMA, to yield an RNAP-promoterclosed complex; (ii) RNAP melts approximately 14 bp of promoter DNAsurrounding the transcription start site, to yield an RNAP-promoter opencomplex; (iii) RNAP begins synthesis of RNA, typically carrying outmultiple rounds of abortive initiation (synthesis and release of RNAproducts less than 9-11 nt in length), as an RNAP-promoter initialtranscribing complex; and (iv), upon synthesis of an RNA product of acritical threshold length of 9-11 nt, RNAP breaks its interactions withpromoter DNA and begins to translocate along DNA, processivelysynthesizing RNA as an RNAP-DNA elongation complex.

To determine whether MccJ25 inhibits steps in transcription up to andincluding formation of the RNAP-promoter open complex, electrophoreticmobility-shift experiments were performed. RNAP holoenzyme was incubatedwith a fluorochrome-labelled DNA fragment containing the lacUV5promoter—in parallel in the absence and presence of MccJ25—and weanalyzed products by non-denaturing PAGE followed by x/y fluorescencescanning (FIG. 3). The results indicate that MccJ25 at a concentrationof 1, 10, or 100 μM has no effect on formation of open complex (FIG. 3).MccJ25 does not inhibit steps in transcription up to and includingformation of open complex.

MccJ25 Inhibits Abortive Initiation and Elongation:

To determine whether MccJ25 inhibits steps in transcription subsequentto formation of the RNAP-promoter open complex, standard transcriptionexperiments were performed. RNAP holoenzyme was pre-incubated with a DNAfragment containing the lacUVS promoter to form the RNAP-promoter opencomplex; radiolabelled NTPs were added and RNA synthesis was allowed toproceed for 5 min at 37° C.—in parallel in the absence and presence ofMccJ25—and products were analyzed by urea-PAGE followed bystorage-phosphor imaging (FIG. 4). The results indicate that MccJ25 at aconcentration of 1, 10, or 100 μM inhibits both formation of abortiveproducts (7 nt and 8 nt RNA species produced in large stoichiometricexcess over the DNA template (see Record et al. 1996; deHaseth et al.1998)) and formation of the full-length product (26 nt RNA species;produced in stoichiometric equivalence with DNA template) (FIG. 4A). Theinhibition is specific; thus, inhibition is overcome completely uponsubstitution of residue 931 of the RNAP β′ subunit—the substitutionshown by Delgado et al., (2001) J. Bacteriol. 183, 4543-4550, to conferresistance to MccJ25 in viva (FIG. 4B). Parallel experiments startingwith a stalled elongation complex, rather than with open complex, yieldequivalent results with respect to inhibition of formation offull-length (unpublished results). MccJ25 inhibits both abortiveinitiation and elongation. MccJ25 interferes with a process common toboth abortive initiation and elongation—i.e., NTP uptake,phosphodiester-bond formation, or translocation.

MccJ25 Inhibits by Interfering with NTP Uptake:

To assess whether inhibition by MccJ25 is NTP-concentration-dependent,transcription experiments were performed at each of four NTPconcentrations (12.5 μM, 25 μM, 50 μM, and 100 μM). The results indicatethat high NTP concentrations can overcome inhibition by MccJ25 (FIG.5A). As the NTP concentration increases, the extent of inhibition byMccJ25 decreases—both at the level of abortive products and at the levelof the full-length product (FIG. 5A). Thus, inhibition by MccJ25 isNTP-concentration-dependent.

Quantitative analysis of the NTP-concentration-dependence data indicatesthat mode of inhibition by MccJ25 is partial competitive—i.e., thatMccJ25 binds to a site on RNAP distinct from the NTP binding site andincreases K_(M) for NTPs (FIG. 5B). K_(i), the reciprocal of theequilibrium binding constant for MccJ25-RNAP interaction, is estimatedto be 1.4±0.2 μM; α, the factor by which MccJ25 increases K_(M) forNTPs, is estimated to be 15±3 (FIG. 5B). Fluorescence-detected abortiveinitiation assays assessing iterative tri- and tetranucleotidesynthesis—assays for which the initial-velocity assumption is rigorouslyvalid—yield equivalent results: i.e., partial-competitive inhibition,with K_(i)=1.2±0.3 μM, and α 8.7±2 (FIG. 5C). MccJ25 inhibitstranscription by binding to a site on RNAP distinct from the NTP bindingsite and interfering with NTP uptake.

MccJ25 Binds Within the RNAP Secondary Channel—Random Mutagenesis:

The MccJ25-resistant rpoC mutant of Delgado et al. 2001 results insubstitution of residue 931 of RNAP β′ subunit (Thr931→Ile). In theprimary structure of β′, residue 931 maps to conserved region G (FIG.6A). In the three-dimensional structure of bacterial RNAP, residue 931of β′ maps to the RNAP NTP-uptake channel (also referred to as thesecondary channel or pore; FIG. 6B). The RNAP secondary channel is a 25Å long ˜10-15 Å wide, tunnel through which NTPs must pass to access theRNAP active-center and NTP binding site (Zhang et al., (1999) Cell 98,811-824; Cramer et al., (2001) Science 292, 1863-1876; and Ebright(2000) J. Mol. Biol. 304, 687-689). The location of the substitution, inconjunction with our finding that MccJ25 inhibits transcription byinterfering with NTP uptake, immediately suggests a possible mechanismof inhibition: i.e., MccJ25 may inhibit transcription by binding within,and obstructing, the RNAP secondary channel.

To define determinants of β′ specifically required for transcriptioninhibition by MccJ25—and thereby to test the hypothesis thatdeterminants for binding of MccJ25 are located within the RNAP secondarychannel-random mutagenesis of the entire gene encoding β′ were performedand isolated and characterized MccJ25-resistant mutants. (RNAP β′subunit comprises nearly one-half of all residues of RNAP and comprisesall residues of the RNAP secondary channel). Mutagenesis usingerror-prone PCR was performed (Zhou (1991) Nucl. Acids Res. 19, 6052;Zhou et al., (1993) Proc. Natl. Acad. Sci. USA 90, 6081-6085) of fiveDNA segments spanning the length of a plasmid-borne rpoC gene (Table 1).Overall, 20 mutagenesis reactions were performed, ˜100,000 candidateswere analyzed, and 22 independent plasmid-linked MccJ25-resistantmutants were isolated (Table 1). Minimum-bacteriocidal-concentration(MBC) assays indicate that all 22 MccJ25-resistant mutants exhibit≧50-fold increases in MBC, and 18 of 22 MccJ25-resistant mutants exhibit≧50-fold increases in MBC (Table 2, column 5). Complementation assaysindicate that all 22 MccJ25-resistant mutants can complement anrpoC^(ts) mutant for growth at the non-permissive temperature,confirming that each encodes a β′ derivative functional intranscription—indeed sufficiently functional in transcription to supportviability (Table 2, column 4).

TABLE 1 Summary of random mutagenesis and selection codons 1-292mutagenesis reactions 4 total candidates 11,000 independentplasmid-linked MccJ25^(r) candidates 0 codons 292-546 mutagenesisreactions 3 total candidates 13,000 independent plasmid-linkedMccJ25^(r) candidates 9 codons 546-876 mutagenesis reactions 4 totalcandidates 11,000 independent plasmid-linked MccJ25^(r) candidates 11codons 876-1213 mutagenesis reactions 5 total candidates 36,000independent plasmid-linked MccJ25^(r) candidates 1 codons 1213-1408mutagenesis reactions 4 total candidates 25,000 independentplasmid-lined MccJ25^(r) candidates 1 overall mutagenesis reactions 20total candidates 96,000 independent plasmid-Linked MccJ25^(r) candidates22

For each of the 22 MccJ25-resistant mutants, the DNA-nucleotide sequenceof the relevant segment of the rpoC gene was determined, and the aminoacid sequence of the substituted β′ derivative was inferred (Table 2).Nineteen different substitutions, involving eighteen different siteswithin β′, were obtained (Table 2).

TABLE 2 MccJ25^(r) isolates from random mutagenesis and selectionNumber of Amino acid Codon independent Complementation MBC* substitutionsubstitution isolates of rpoC^(ts) (mg/ml) none none — ++ 0.01 424 Asn→Ser AAC→AGC   1** + 0.1  428 Thr→Ile ACT→ATT 2 ++ 0.5 430 His→Leu CAC→CTC 3 + 0.5  464 Asp→Gly GAC→GCC 1 ++ 0.05  469 His→ArgCAC→CGC 1 ++ 0.05  504 Gln→Arg CAG→CGG 1 ++ 0.5  733 Ser→Phe TCT→TTC 1 +0.5  738 Arg→Leu CGT→CTT 1 ++ 0.05  776 Thr→Ile ACC→ATC 1 ++ 0.5 779 Ala→Thr GCT→ACT 1 ++ 1  780 Arg→Cys CGT→TGT 1 ++ 1  782 Gly→AlaGGT→GCT 1 ++ 1  785 Asp→Gly GAT→GGT 1 ++ 0.5  786 Thr→Ile ACC→ATC 1 ++0.5  789 Lys→Arg AAA→AGA 1 ++ 0.5  789 Lys→Gln AAA→CAA 1 ++ 1 869 Cys→Arg TGT→CGT 1 + 1  933 Arg→Cys CGT→TGT 1 ++ 1 1244 Gln→LeuCAG→CTG 1 ++ 0.5 *Minimum bacteriocidal concentration; defined as thelowest concentration of MccJ25 that yields a viable cell count of ^(~)0after incubation 2 h at 37° C. (see Exeperimental Procedures).**Isolated as double mutant 354 Val→Ile; 424 Asn→Ser; complementationand MBC data are for a single mutant constructed using site-directedmutagenesis.

In the primary structure of β′, the sites at which substitutions wereobtained map to conserved region D, conserved region E, conserved regionF, the segment between conserved regions F and G, conserved region G,and the segment between conserved regions G and H (referred to asconserved region G′ by Zakharova et Al., (1998) J. Biol. Chem. 273,24912-24920) (FIG. 6A). In the three-dimensional structure of RNAP, thelocations of the substitutions are tightly clustered—and are centered onthe RNAP secondary channel (FIG. 6C). The substitutions, withoutexception, map to residues that line the RNAP secondary channel, or toresidues that make direct contact with residues that line the RNAPSecondary channel (FIG. 6C). The substitutions map to the floor, theroof, and the walls of RNAP secondary channel (FIG. 6C). The RNAPsecondary channel contains a multi-residue determinant for function ofMccJ25. Based on the fact that substitutions conferringMccJ25-resistance were obtained at none of the >1000 residues of β′outside the immediate vicinity of the secondary channel, it is concludedthat no part of β′ outside the immediate vicinity of the secondarychannel contains a determinant for function of MccJ255.

MccJ25 Binds within the RNAP Secondary Channel—Saturation Mutagenesis:

To define systematically the MccJ25 determinant within the RNAPsecondary channel, saturation mutagenesis of the rpoC gene, wasperformed, targeting all codons for residues that line the RNAPsecondary channel. A saturation mutagenesis using a set often “doped”oligodeoxyribonucleotide primers, designed to introduce all possiblenucleotide substitutions at all positions of all codons for residuesthat line the RNAP secondary channel was performed (sequences in Table3; methods essentially as in the following references (Ner et al.,(1988) DNA 7, 127-134; Hermes et al., (1989) Gene 84, 143-151; and Niuet al., (1994) J. Mol. Biol. 243, 595-602). In total, 23 mutagenesisreactions were performed, ˜40,000 candidates were analyzed, and 105independent plasmid-linked MccJ25-resistant mutants were isolated andcharacterized (Table 4).

TABLE 3 Sequences of “doped” oligonucleotide primers used insaturation mutagenesis Codon Sequence  429-433*CCGTGCACCGACTCTGCACCGTCTGGGTATCCAGGCATTTG (SEQ ID NO: 26)  492-504**GCAACAACATCCTGTCCCCGGCGAACGGCGAACCAATCATCGTTCCGTCTCAGG ACGTTGTACTGGGTC(SEQ ID NO: 27)  597-603*GTCAACCAGGCGCTGGGTAAAAAAGCAATCTCCAAAATGCTGAACACCTGC (SEQ ID NO: 28) 680-698** CGGGCGAACGCTACAACAAAGTTATCGATATCTGGGCTGCGGCGAACGATCGTGTATCCAAAGCGATGATGGATAACCTGCAAAC (SEQ ID NO: 29)  726-740**CTACATGATGGCCGACTCCGGTGCGCGTGGTTCTGCGGCACAGATTCGTCAGCTT GCTGGTATG(SEQ ID NO: 30)  741-754**CGTCAGCTTGCTGGTATGCGTGGTCTGATGGCGAAGCCGGATGGCTCCATC ATCGAAACG(SEQ ID NO: 31)  775-790**GTACTTCATCTCCACCCACGGTGCTCGTAAAGGTCTGGCGGATACCGCACTGAAAA CTGCGAACTCCG(SEQ ID NO: 32)  922-933**GTGTTATCGCGGGACAGTCCATCGGTGAACCGGGTACACAGCTGACCATGCGTACG TTCCACATCCGTGG(SEQ ID NO: 33) 1136-1137* CCAAGGACATCACCGGTGGTCTGCCGCGCGTTGC(SEQ ID NO: 34) 1239-1248**CGAAGTACAGGACGTATACCGTGTGCAGGCCGTTAAGATTAACGATAAAC (SEQ ID NO: 35)*Underlined nucleotides were synthesized using phosphoramiditereservoirs having 92% correct phosphoramidite and 8% of a 1:1:1:1 mix ofdA, dC, dG, and dT phosphoramidites (i.e., 94% of total correctphosphoramidites and 6% of total incorrect phosphoramidites).**Underlined nucleotides were synthesized using phosphoramiditereservoirs having 98% correct phosphoramidite and 2% of a 1:1:1:1 mix ofdA, dC, dG, and dT phosphoramidites (i.e., 98.5% of total correctphosphoramidites and 1.5% of total incorrect phosphoramidites).

Sequencing indicates that 100 of the 105 MccJ25-resistant mutants aresingle-substitution mutants (Table 4). The single-substitution mutantscomprise 71 different substitutions, involving 43 different sites withinβ′ (Table 4).

TABLE 4 Summary of saturation mutagenesis and selection codons 429-433mutagenesis reactions 4 total candidates 6,000 independentplasmid-linked MccJ25^(r) candidates 8 codons 492-504 mutagenesisreactions 2 total candidates 2,000 independent plasmid-linked MccJ25^(r)candidates 9 codons 597-603 mutagenesis reactions 3 total candidates5,000 independent plasmid-linked MccJ25^(r) candidates 0 codons 680-698mutagenesis reactions 2 total candidates 8,000 independentplasmid-linked MccJ25^(r) candidates 6 codons 726-740 mutagenesisreactions 2 total candidates 3,000 independent plasmid-linked MccJ25^(r)candidates 12 codons 741-754 mutagenesis reactions 2 total candidates4,000 independent plasmid-linked MccJ25^(r) candidates 5 codons 775-790mutagenesis reactions 3 total candidates 3,000 independentplasmid-linked MccJ25^(r) candidates 33 codons 922-933 mutagenesisreactions 2 total candidates 1,000 independent plasmid-linked MccJ25^(r)candidates 13 codons 1136-1137 mutagenesis reactions 1 total candidates2,000 independent plasmid-linked MccJ25^(r) candidates 4 codons1239-1248 mutagenesis reactions 2 total candidates 3,000 independentplasmid-linked MccJ25^(r) candidates 15 overall mutagenesis reactions 23total candidates 37,000 independent plasmid-linked MccJ25^(r) candidates105

In the three-dimensional structure of RNAP, the sites at which singlesubstitutions were obtained define a nearly continuous surface,comprising most of the interior lining and part of the rim of the RNAPsecondary channel (FIG. 6D). The sites span nearly the fullcircumference of the RNAP secondary channel (FIG. 6D). The side chainsof the majority of implicated residues are solvent-accessible—directedinto the lumen of the RNAP secondary channel or toward the exterior ofRNAP—and make no obvious interactions important for RNAP structure orfunction.

The present invention provides that the RNAP secondary channel containsan extensive determinant for function of MccJ25. Based on the size ofthe determinant (nearly 50 residues; Tables 3 and 6), the architectureof the determinant (interior of hollow cylinder), and thesolvent-accessibility of the determinant. The present invention providesthat the determinant corresponds to the binding site on RNAP for MccJ25.For reference, the size and molecular mass of MccJ25 (2,107 Da;diameter=19 Å, modeled as a sphere) would allow MccJ25 to make directcontact with all, or nearly all, residues of the determinant. Thepresent invention also provides that the sites of substitutions thatconfer MccJ25-resistance map the binding site on RNAP for MccJ25 and, inessence, serve as a genetic footprint of the binding site.

TABLE 5 MccJ25^(r) isolates from saturation mutagenesis and selectionNumber of Amino acid independent MBC* substitution isolates (mg/mi)Single-substitution mutants  428 Thr->Ile 3 0.5  428 Thr->Asn 2 0.5  429Leu->Gln 2 0.5  430 His->Tyr 1 0.5  498 Pro->Leu 1 1  498 Pro->Gln 1 1 503 Ser->Pro 1 0.5  503 Ser->Tyr 1 0.5  504 Gln->Arg 1 0.5  504Gln->Glu 2 0.5  508 Leu->Val 1 0.05  680 Asn->Lys 2 0.5  684 Asp->Ala 11  684 Asp->Tyr 1 0.5  684 Asp->Glu 1 0.5  684 Asp->Vai 1 0.5  732Gly->Asp 2 0.1  733 Ser->Phe 1 0.1  733 Ser->Val 1 0.5  733 Ser->Tyr 10.1  735 Ala->Δ 2 1  736 Gln->Pro 1 0.5  738 Arg->Leu 1 0.05  744Arg->Pro 1 0.05  744 Arg->His 1 0.05  748 Ala->Pro 3 0.1  775 Ser->Cys 10.5  776 Thr->Ile 3 0.5  777 His->Tyr 1 1  777 His->Pro 2 0.5  779Ala->Gly 1 0.5  779 Ala->Thr 1 1  779 Ala->Pro 2 0.5  780 Arg->Cys 1 1 782 Gly->Ala 1 1  783 Leu->Pro 1 0.1  784 Ala->Glu 1 0.5  785 Asp->Gly2 0.5  786 Thr->Ile 1 0.5  786 Thr->Ala 2 0.5  788 Leu->Met 1 1  789Lys->Asn 2 1  789 Lys->Gln 1 1  789 Lys->Arg 1 0.5  790 Thr->Ala 2 1 790 Thr->Ile 2 1  790 Thr->Ser 2 0.5  790 Thr->Asn 1 0.5  922 Cys->Tyr1 0.5  926 Pro->Ser 1 0.5  927 Gly->Ser 2 1  927 Gly->Cys 1 1  930Leu->Met 1 1  931 Thr->Ile 2 1  931 Thr->Ala 2 1  932 Met->Ile 2 1  933Arg->Cys 1 1 1136 Gly->Cys 1 1 1136 Gly->Ala 1 0.5 1137 Gly->Aia 1 0.51137 Gly->Arg 1 1 1240 Val->Glu 1 0.5 1241 Tyr->Ser 2 0.1 1241 Tyr->His2 0.1 1241 Tyr->Cys 1 0.1 1244 Gln->Pro 2 0.1 1244 Gln->Leu 1 0.5 1244Gln->Glu 2 0.1 1247 Lys->Glu 2 0.1 1247 Lys->Gln 1 0.1 1248 lle->Ser 10.1 multiple-substitution mutants  493 Pro->Thr 1 0.1  498 Pro->Thr  732Gly->Asp 1 0.1  733 Ser->Ala  732 Gly->Asp 1 0.5  735 Gly->Thr  733Ser->Val 1 0.1  734 Ala->Gly  777 His->Ser 1 0.5  778 Gly->Ala Minimumbacteriocidal concentration; defined as the lowest concentration ofMccJ25 that yields a viable cell count of ~0 after incubation 2 hr at37° C. (see Experimental Procedures).

Thirteen of fifteen sites associated with the highest level ofMccJ25-resistance (MBC=1 mg/ml; Tables 2 and 5) cluster in an ˜20 Å×˜20Å×˜20 Å sub-region of the RNAP secondary channel, bounded by the α-helixcontaining residue 684 (D/E helix), the α-helix containing residue 735(E helix), the α-helix containing residues 777-790 (F helix), theα-helix and loop containing residues 927-933 (G helix and loop), andloop containing residues 1136-1137 (G′ loop) (sub-region above and toleft of the active-center Mg⁺⁺ in view in FIG. 6D). The presentinvention provides that this sub-region is the most important part ofthe determinant.

Single substitutions at five sites within β′ confer MccJ25-resistance:733, 783, 931, 935, and 1138 (Yuzenkova et al., (2002) J. Biol. Chem.277, 50867-50875). The target for MccJ25 includes these sites within β′(four of which are reported herein; Tables 2 and 5).

The present invention provides that MccJ25 inhibits theabortive-initiation and elongation phases of transcription, thatinhibition involves interference with NTP uptake, that inhibition ispartial-competitive with respect to NTPs (i.e., involves a site distinctfrom the RNAP NTP binding site), and that inhibition involves anextensive determinant within the RNAP secondary channel, comprisingnearly the entire lining of the RNAP secondary channel (nearly fiftysites for substitutions conferring MccJ25-resistance). The results ofYuzenkova et al., (2002) J. Biol. Chem. 277, 50867-50875, althoughlimited, also indicate that inhibition involves a multi-residuedeterminant within the RNAP secondary channel (five sites forsubstitutions conferring MccJ25-resistance). Preliminary results offluorescence-resonance-energy-transfer experiments with afluorochrome-labelled MccJ25 derivative provide direct evidence thatMccJ25 binds within the RNAP secondary channel (methods as in Mekler etal., (2002) Cell 108, 599-614). The RNAP secondary channel is a 25 Ålong, ˜10-15 Å wide, fully enclosed tunnel through which NTPs must passto access the RNAP active-center and NTP binding site (Zhang et al.,(1999) Cell 98, 811-824; Cramer et al., (2001) Science 292, 1863-1876;and Ebright (2000) J. Mol. Biol. 304, 687-689). MccJ25 inhibitstranscription by binding within, and obstructing, the RNAP secondarychannel—acting essentially as a “cork in a bottle.”

The present invention also provides that a molecule the size of MccJ25(2.1 kDa; diameter=19 Å, modeled as a sphere) readily could bind within,and obstruct, the RNAP secondary channel (dimensions=25 Å by 10-15 Å).

Obstruction of the RNAP secondary channel represents a novel mechanismof inhibition of RNAP. Rifampicin, an inhibitor of bacterial RNAP,functions by sterically preventing synthesis of an RNA product longerthan ˜4 nt (Campbell et al., (2001) Cell 104, 901-912). Streptolydiginand α-amanitin, inhibitors of bacterial RNAP and eukaryotic RNAP II,respectively, are proposed to Interfere with active-centerconformational changes associated with phosphodiester-bond formationand/or translocation (Epshtein et al., (2002) Mol. Cell 10, 623-634;Bushnell et al., (2002) Proc. Natl. Acad. Sci. USA 99, 1218-1222).

Several arguments suggest that obstruction of the RNAP secondary channelrepresents an exceptionally attractive target for development of novelantimicrobial agents. First, the RNAP secondary channel is eminently“druggable,” presenting an extended, encircling surface complementary toa range of molecules—like MccJ25—that have molecular weights of500-2,500 Da. Second, the RNAP secondary channel exhibits distinctpatterns of sequence conservation in bacterial RNAP and eukaryotic RNAP,permitting identification of agents—like MccJ25—that inhibit bacterialRNAP but do not inhibit eukaryotic RNAP. Third, the RNAP secondarychannel is distinct from, and well-separated from, the binding site ofthe RNAP inhibitor in current use in antimicrobial therapy, rifampicin,permitting identification of agents—like MccJ25 (unpublished data)—thatdo not exhibit cross-resistance with rifampicin.

Experimental Procedures:

Plasmids:

Plasmid pTUC202 carries genes for synthesis and export of MccJ25(Solbiati et al., (1999) J. Bacterial. 181, 2659-2662; gift of Dr. F.Moreno). Plasmid pRL663 encodes C-terminally hexahistidine-tagged E.coli RNAP β′ subunit under control of the tac promoter (Wang et al.,(1995) Cell 81, 341-350; gift of Dr. R. Landick). Plasmid pREII-NHαencodes N-terminally hexahistidine-tagged E. coli RNAP α subunit undercontrol of tandem lpp and 'lacUV5 promoters (Niu et al., 1996).

MccJ25:

MccJ25 was purified essentially as in Blond et al. 1999. E. coli strainDH5α (hsdR17 recA1 relA1 endA1 gyrA96 gal deoR phoA supE44 thi Δ(lacZYA-argF) U169ø80dlacZΔM15; Invitrogen, Inc). transformed withplasmid pTUC202 (Solbiati et al., 1996) was shaken in 1 L M9 medium(Sambrook and Russell (2001). Molecular Cloning; A Laboratory Manual(Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory)), for 24 h at37° C., and the culture medium was harvested by centrifugation (5000×g;15 mm at 22° C.). The culture medium was applied to a preparative C8cartridge (10 g Mega-BE C8; Varian, Inc); the cartridge was washedsuccessively with 50 ml water, 50 ml 20% methanol, and 50 ml 50%methanol; the cartridge was eluted with 4×5 ml fractions of 80%methanol; and fractions containing MccJ25 (detected by UV absorbance at276 nm) were pooled, lyophilized, re-dissolved in 10 ml 20% methanol,and stored in aliquots at −20° C. Yields typically were 100 mg(determined by UV-absorbance at 276 nm using ε₂₇₆=2,900 M⁻¹ cm⁻¹(calculated as in Gill and von Hippel, 1989 (1991) J. Mol. Biol. 220,307-324)). Purities typically were 50% (determined by analyticalreversed-phase HPLC). Samples for electrophoretic mobility shift assays,transcription assays, and fluorescence-detected abortive initiationassays were further purified by reversed-phase HPLC on a C18, 5 μM, 300Å column (Rainin, Inc)., with solvent A=20% methanol and 0.1%trifluoroacetic acid, solvent B=methanol and 0.1% trifluoroacetic acid,and gradient=0-90% solvent B in solvent A in 50 mm, flow rate=1 ml/min.Fractions containing MccJ25 (retention time ˜30 mm; detected by UVabsorbance at 276 nm) were pooled, lyophilized, re-dissolved in 500 μl10% methanol, and stored in aliquots at −20° C. Yields typically were 50mg per 1 L culture. Purities typically were >95%.

RNAP:

RNAP holoenzyme was prepared from, strain XE54/pREII-NHα (thi pREII-NHα;Niu et al., (1996) Cell 87, 1123-1134) using metal-ion affinitychromatography and ion-exchange chromatography (Id.). (Ile931]β′-RNAPholoenzyme was prepared from strain 397c [rpoC^(ts)397 argG thi lacλcI₈₅₇h₈₀S_(t68)dlac⁺); Christie et al., (1996) J. Bacteriol. 178,6991-6993) transformed with a pRL663 derivative encoding [Ile931]β′-RNAP(constructed using site-directed mutagenesis (QuikChange Site-DirectedMutagenesis Kit; Stratagene, Inc).) using identical procedures. Yieldstypically were 6 mg. Purities typically were >95%.

Electrophoretic Mobility Shift Assays:

Reaction mixtures contained (20 μl): 100 nM RNAP holoenzyme, 20 nM DNAfragment lacUV5-12(Cy5,+26) (Mukhopadhyay et al., (2001) Cell 106,453-463), and 0-100 μM MccJ25 in TB [50 mM Tris-HCl (pH 8.0), 100 mMKCI, 10 mM MgCl₂, 1 mM dithiothreitol, 10 μg/ml bovine serum albumin,and 5% glycerol]. Following 15 min at 37° C., 0.5 μl 1 mg/ml heparin wasadded (to disrupt non-specific complexes (Cech and McClure (1980)Biochem. 19, 2440-2447)), and, following a further 2 min at 37° C.,reaction mixtures were applied to 5% polyacrylamide slab gels (30:1acrylamide/bisacrylamide; 6×9×0.1 cm) and electrophoresed in 90 mMTris-borate (pH 8.0) and 0.2 mM EDTA (20 V/cm; 30 mm at 37° C.) andanalyzed using an x/y fluorescence scanner (Storm 860; MolecularDynamics, Inc.).

Transcription Assays:

Reaction mixtures contained (18 μl): 100 nM RNAP holoenzyme, 20 nM DNAfragment lacUV5-12(Cy5,+26) (Mukhopadhyay et al., (2001) Cell 106,453-463), and 0-100 μM MccJ25 in TB. Following 15 min at 37° C., 0.5 μl1 mg/ml heparin, was added, and, following a further 2 min at 37° C.,RNA synthesis was initiated by addition of 2 μl 5 mM ApA and 125 μM (or250 jiM, 500 μM, and 1 mM) each of [α-³²P]UTP (0.6 Bq/fmol), ATP, CTP,GTP. Following 5 min at 37° C., reactions were terminated by addition of10 μl 80% formamide, 10 mM EDTA, 0.04% bromophenol blue, and 0.04%xylene cyanol. Products were heated 10 min at 90° C., resolved byurea-PAGE (Sambrook and Russell (2001). Molecular Cloning: A LaboratoryManual (Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory)), andquantified using an x/y storage-phosphor scanner (Storm 860; MolecularDynamics, Inc.). Data were fit to full-competitive, partial-competitive,full-noncompetitive, partial-noncompetitive, full-uncompetitive,partial-uncompetitive, full-mixed, and partial-mixed models ofinhibition using the Fit-To-Model feature of the SigmaPlot EnzymeKinetics Module (SPSS, Inc.).

Fluorescence-detected Abortive Initiation Assays:

Reaction mixtures contained (46.5 μl): 100 nM RNAP holoenzyme, 20 nM DNAfragment lacUV5-12 (Mukhopadhyay et al., (2001) Cell 106, 453-463), and0-100 μM MccJ25 in TB. Following 15 min at 37° C. 0.5 μl mg/ml heparinwas added, and, following a further 2 min at 37° C., 1 μl of 0.25-5 mM(y-AmNS)UTP (Molecular Probes, Inc.) was added, and reaction mixtureswere transferred to sub-micro fluorometer cuvettes (Starna Cells, Inc.).Following 2 min at 37° C., RNA synthesis was initiated by addition of2.5 μl 10 mM A_(p)A, and fluorescence emission intensity was monitored 5min at 37° C. [excitation wavelength=360 nm and emission wavelength=500nm; excitation and emission slit widths=4 nm; QuantaMaster QM1spectrofluororaeter (PTI, Inc).]. The quantity of UMP incorporated intoRINA was determined from the quantity of (γ−AmNS)UTP consumed, which, inturn, was calculated as follows (Schlageck et al. 1979):(γ−AmNS)UTP _(consumed)=[(γ−AmNS)UTP ₀](F _(t) −F ₀)/(12.4×F ₀)where (γ−AmNS)UTP₀ is the quantity of (γ−AmNS)UTP at time 0, F₀ is thefluorescence emission intensity at time 0, and F_(t) is the fluorescenceemission intensity at time t. Data were fit to full-competitive,partial-competitive, full-noncompetitive, partial-noncompetitive,full-uncompetitive, partial-uncompetitive, full-mixed, and partial-mixedmodels of inhibition as in the preceding section.Random Mutagenesis:

Random mutagenesis was performed by error-prone PCR amplification of theXbaI-SnaBI (codons 1-292), SnaBI-SphI (codons 292-546), SphI-SalI(codons 546-876), SalI-BspEI (codons 876-1213), and BspEI-XhoI (codons1213-1408) rpoC segments of plasmid pRL663 (procedure of Zhou (1991)Nucl. Acids Res. 19, 6052; Zhou et al., (1993) Proc. Natl. Acad. Sci.USA 90, 6081-6085). The mutagenesis procedure yields all possibletransition and transversion substitutions (Id. at Table 2). Mutagenizedplasmid DNA was introduced by transformation into strain Stbl2 [mcrAΔ(mcrBC-hsdRMS-mrr) recA1 relA1 endA1 gyrA96 lon supE44 thiΔ(lac-proAB); Invitrogen, Inc.], transformants (−10⁴ cells) were appliedto LB-agar plates (Sambrook and Russell (2001). Molecular Cloning: ALaboratory Manual (Cold Spring Harbor, N.Y., Cold Spring HarborLaboratory)) containing 1 μg/ml MccJ25 and 200 μg/ml ampicillin, andplates were incubated 24 h at 37° C. followed by 0-48 h at 25° C. Foreach MccJ25^(r) clone (identified as a clone yielding a colony on theoriginal selective plate and also yielding colonies when re-streaked tothe same medium and incubated 16 h at 37° C.), plasmid DNA was prepared,plasmid DNA was introduced by transformation into strain DH5α [hsdR17recA1 relA1 endA1 gyzA96 gal deoR phoA supE44 thi Δ(lacZYA-argF)U169ø80dlacZΔM15; Invitrogen, Inc.], transformants (˜10⁴ cells) wereapplied to LB-agar plates containing I μg/ml MccJ25 and 200 μg/mlampicillin and, in parallel, to LB-agar plates containing 200 μg/mlampicillin, and plates were incubated 16 h at 32° C. For eachplasmid-linked MccJ25r clone (identified as a clone yielding comparablenumbers of colonies on the plates with and without MccJ25), thenucleotide sequence of the mutagenized rpoC segment was determined bydideoxy nucleotide sequencing.

Saturation Mutagenesis:

A set of “doped” oligodeoxyribonucleotide primers corresponding tocodons 425-437, 487-509, 592-608, 675-703, 723-743, 739-757, 772-793,918-937, 1132-1141, and 1236-1251 of the rpoC gene of pRL663 wassynthesized on an AB392 automated synthesizer (Applied Biosystems, Inc).using solid-phase β-cyanoethylphosphoramidite chemistry (sequences inTable 4). The level of “doping” (nucleotide misincorporation) wasselected to yield an average of 0.4-1 substitution per molecule ofoligodeoxyribonucleotide primer (equations in Hermes et al., 1989,1990). Thus, the nucleotides corresponding to codons 429-433, 597-603,and 1136-1137 were synthesized using phosphoramidite reservoirscontaining 92% of the correct phosphoramidite and 8% of a 1:1:1:1 mix ofdA, dC, dG, and dT phosphoramidites (i.e., 94% total correctphosphoramidite and 6% total incorrect phosphoramidite); the nucleotidescorresponding to codons 492-504, 680-698, 726-740, 741-754, 775-790,922-933, and 1239-1248 were synthesized using phosphoramidite reservoirscontaining 98% of the correct phosphoramidite and 2% of a 1:1:1:1 mix ofdA, dC, dG, and dT phosphoramidites (i.e., 98.5% total correctphosphoramidite and 1.5% total incorrect phosphoramidite); and all othernucleotides were synthesized using phosphoramidite reservoirs containing100% of the correct phosphoramidite. Primer-extension mutagenesisreactions were performed using the QuikChange Site-Directed MutagenesisKit (Stratagene, Inc)., with a “doped” oligodeoxyribonucleotide primer,a complementary wild-type oligodeoxyribonucleotide primer, and pRL663 asTemplate (primers at 75 nM; all other components at concentrations asspecified by the manufacturer). Mutagenized plasmid DNA was introducedinto cells, and plasmid-linked MccJ25^(r) clones were identified andcharacterized, as in the preceding section.

Complementation Assays:

Strain 397c [rpoC^(ts) 397 argG thi lac (λcI₈₅₇h₈₀S_(t68)dlac+);Christie et al., 1996] was transformed with pRL663 or a pRL663derivative, transformants (˜10⁴ cells) were applied to LB-agar plates(Sambrook and Russell (2001). Molecular Cloning: A Laboratory Manual(Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory)) containing 1μg/ml MccJ25 and 200 μg/ml ampicillin, plates were incubated 16 h at 37°C., and bacterial growth was scored.

Minimum Bacteriocidal Concentration (Mbc) Assays:

Strain DH5α [hsdR17 recA1 relA1 endA1 gyrA96 gal deoR phoA supE44 thiΔ(lacZYA-argF) U169 ø80dlacZΔM15; Invitrogen, Inc.] was transformed withpRL663 or a pRL663 derivative, transformants (10⁶ cells) were incubated2 h at 37° C. in 1 ml LB (Id.) containing 0.01, 0.05, 0.1, or 1 mg/mlMccJ25; aliquots (10 μl) were applied to LB-agar plates (Id), containing200 μg/ml ampicillin; plates were incubated 16 h at 37° C.; and colonieswere counted. The MBC was defined as the lowest concentration of MccJ25that yielded a colony count of <5.

Example 2 Probe-labelled Derivatives of Peptide Antibiotic MccJ25

One aspect of the invention provides probe-labelled derivatives of thepeptide antibiotic MccJ25 (MccJ25). The invention has broad applicationsin analysis of RNAP structure and function, control of bacterial geneexpression, control of bacterial growth, antibacterial chemistry,antibacterial therapy, and drug discovery.

The present invention also provides a composition comprising a compoundaccording to the general structural formula (I): J-Z—X, wherein J isMccJ25 or a substituted and/or truncated derivative thereof, Z is acovalent linker or is absent, and X is a detectable group. One aspect ofthe present invention, provides a composition comprising a compoundaccording to the general structural formula (I) wherein X is selectedfrom the group consisting of a fluorescent moiety, a phosphorescentmoiety, a luminescent moiety, an absorbent moiety, a photosensitizer, aspin label, a radioisotope, an isotope detectable by nuclear magneticresonance, a paramagnetic atom, a heavy atom, a hapten, a crosslinkingagent, a cleavage agent, and combinations thereof. Another aspect of thepresent invention, provides a composition comprising formula (I) whereinX is a fluorescent moiety. Another aspect of the present invention,provides a composition comprising formula (I) wherein X is a cyaninedye. Another aspect of the present invention, provides a compositioncomprising formula (I) wherein X is a Cy3. Another aspect of the presentinvention, provides a composition comprising formula (I) wherein X is aCy5.

The present invention also provides a composition comprising aderivative of MccJ25 having a detectable group incorporated at position13, according to the general structural formula (II): [O₁₃—Z—X]J,wherein O is an amino acid or amino acid derivative, Z is a covalentlinker or is absent, and X is a detectable group. One aspect of thepresent invention, provides a composition according to formula) (II)wherein O is lysine; and wherein X is selected from the group consistingof a fluorescent moiety, a phosphorescent moiety, a luminescent moiety,an absorbent moiety, a photosensitizer, a spin label, a radioisotope, anIsotope detectable by nuclear magnetic resonance, a paramagnetic atom, aheavy atom, a hapten, a crosslinking agent, a cleavage agent, andcombinations thereof. A preferred aspect of the present invention,provides a composition according to formula (II) wherein X is afluorescent moiety. An especially preferred aspect of the presentinvention, provides a composition according to formula (II) wherein X isa cyanine dye. An especially preferred aspect of the present inventionprovides a composition according to said formula (II) wherein X is Cy3or Cy5.

The present invention also provides a composition comprising aderivative of MccJ25 having a detectable group incorporated at position15, according to the general structural formula (III): [O₁₅—Z—X]J,wherein O is an amino acid or amino acid derivative, Z is a covalentlinker or is absent, and X is a detectable group. One aspect of thepresent invention, provides a composition according to formula) (III)wherein O is lysine; and wherein X is selected from the group consistingof a fluorescent moiety, a phosphorescent moiety, a luminescent moiety,an absorbent moiety, a photosensitizer, a spin label, a radioisotope, anisotope detectable by nuclear magnetic resonance, a paramagnetic atom, aheavy atom, a hapten, a crosslinking agent, a cleavage agent, andcombinations thereof. A preferred aspect of the present inventionprovides a composition according to formula (III) wherein X is afluorescent moiety. An especially preferred aspect of the presentinvention provides a composition according to formula (III) wherein X isa cyanine dye. An especially preferred aspect of the present inventionprovides a composition according to said formula (III) wherein X is Cy3or Cy5.

The present invention also provides a composition comprising aderivative of MccJ25 having a detectable group incorporated at position17, according to the general structural formula (IV): [O₁₇—Z—X]J,wherein O is an amino acid or amino acid derivative, Z is a covalentlinker or is absent, and X is a detectable group. One aspect of thepresent invention, provides a composition according to formula) (IV)wherein O is lysine; and wherein X is selected from the group consistingof a fluorescent moiety, a phosphorescent moiety, a luminescent moiety,an absorbent moiety, a photosensitizer, a spin label, a radioisotope, anisotope detectable by nuclear magnetic resonance, a paramagnetic atom, aheavy atom, a hapten, a crosslinking agent, a cleavage agent, andcombinations thereof. A preferred aspect of the present invention,provides a composition according to formula (IV) wherein X is afluorescent moiety. An especially preferred aspect of the presentinvention provides a composition according to formula (IV) wherein X isa cyanine dye. An especially preferred aspect of the present inventionprovides a composition according to said formula (IV) wherein X is Cy3or Cy5.

The present invention also provides a composition comprising a compoundaccording to any one of the general structural formulas (I), (II),(III), or (IV) for a) analysis, synthesis, screening, or design ofligands of RNAP; b) analysis, synthesis, screening, or design ofmodulators of RNAP activity; c) analysis, synthesis, screening, ordesign of modulators of bacterial gene expression; and d) analysis,synthesis, screening, or design of modulators of bacterial growth.

In a preferred embodiment, the invention provides for the use of acompound according to any one of the general structural formulas (I),(II), (III), or (IV) in an assay assessing the ability of a molecule, orset of molecules, to displace said compound from a bacterial RNAP, or afragment thereof, or to compete with said compound for binding to abacterial RNAP, or a fragment thereof.

In an especially preferred embodiment, the invention provides for theuse of a compound according to any one of the general structuralformulas (I), (II), (III), or (IV) in a homogeneous assay FRET assaymeasuring the ability of a molecule, or set of molecules, to displacesaid compound from a bacterial RNAP, or a fragment thereof, or tocompete with said compound for binding to a bacterial RNAP, or afragment thereof.

The present invention provides for the preparation of [Cy3-Lys13]MccJ25(a compound according to structural formulas (I) and (II)) anddemonstration that the compound binds to and inhibits RNAP with highpotency. The present invention also provides for the preparation of[Lys13]MccJ25, [Lys15]MccJ25, and [Lys17]MccJ25 (intermediates insynthesis of compounds according to structural formulas (I)-(IV) anddemonstration that the compounds bind to and inhibit RNAP with highpotency.

The present invention also provides for the use of Cy3-Lys13]MccJ25 (acompound according to structural formulas (I) and (II)) in a homogeneousFRET assay measuring the ability of a molecule, or set of molecules, todisplace said compound from a bacterial RNAP, or a fragment thereof, orto compete with said compound for binding to a bacterial RNAP, or afragment thereof.

INDUSTRIAL APPLICABILITY

The invention has applications in control of bacterial gene expression,control of bacterial growth, antibacterial chemistry, and antibacterialtherapy.

Although the invention herein has been described with reference toparticular embodiments, it is to be understood that these embodimentsare merely illustrative of the principles and applications of thepresent invention. It is therefore to be understood that numerousmodifications may be made to the illustrative embodiments and that otherarrangements may be devised without departing from the spirit and scopeof the present invention as defined by the appended claims.

All patent and non-patent publications cited In this specification areindicative of the level of skill of those skilled In the art to whichthis invention pertains. All these publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated herein by reference.

The invention claimed is:
 1. An isolated analog of bacteriocidal-peptidemicrocin J25 (MccJ25) that (1) has an amino acid sequence that differsfrom that of MccJ25 in terms of at least one amino acid substitution,insertion, or deletion; and (2) that binds a bacterial RNAP and inhibitsan activity of bacterial RNAP with a potency at least equal to that ofMccJ25 wherein the analog is selected from the group consisting of[Lys₅]MccJ25, [Lys₁₃]MccJ25, [Lys₁₅]MccJ25, and [Lys₁₇]MccJ25.
 2. Theisolated analog according to claim 1 that also contains a detectablegroup.
 3. The isolated analog according to claim 2 wherein thedetectable group is selected from the group consisting of a chromophore,fluorophore and Cy3.
 4. An isolated analog of bacteriocidal-peptidemicrocin J 25 (MccJ25) that (1) has an amino acid sequence that differsfrom that of MccJ25 in terms of at least one amino acid substitution,insertion, or deletion; and (2) that binds a bacterial RNAP and inhibitsan activity of bacterial RNAP with a potency at least equal to that ofMccJ 25 wherein the analog is selected from the group consisting of[X-Lys₅]MccJ25, [X-Lys₁₃]MccJ25, [X-Lys₁₅]MccJ25, and [X-Lys₁₇]MccJ25,wherein X is a detectable group.
 5. The isolated analog according toclaim 4 wherein the detectable group is selected from the groupconsisting of a chromophore, fluorophore and Cy3.